Skip to main content
Download PDF
  • Research article
  • Open access
  • Published:

Impact of sodium alginate hydrogel containing bacteriophage peptides that specifically bind to the EtCab protein on the inhibition of Eimeria tenella infection

Veterinary Research volume 56, Article number: 18 (2025) Cite this article

Abstract

Avian coccidiosis, caused by the protozoan Eimeria, leads to significant economic losses for the poultry industry. In this study, bacteriophages that specifically bind to the calcium-binding protein (EtCab) of Eimeria tenella were selected using a biopanning process with a pIII phage display library. The recombinant EtCab protein served as the ligand in this selection process. The binding ability of target phages to the EtCab protein or E. tenella sporozoites was evaluated. The role of peptides corresponding to target phages in inhibiting the invasion of E. tenella sporozoites into cells was analysed using flow cytometry. Subsequently, the phages were encapsulated in sodium alginate to protect them from degradation in gastric fluid, which has a low pH value. Chickens were orally administered both microencapsulated and non-microencapsulated phages, and the protective effects against E. tenella infection were assessed. The binding mechanism of these peptides to the EtCab protein was investigated through in silico analysis. The results indicated that three specific phages (Y, G, and V) could bind effectively to recombinant EtCab protein as well as to sporozoite proteins. All three peptides, particularly Y and G, demonstrated significant inhibition of sporozoite invasion into cells in vitro. Additionally, oral administration of the encapsulated phages Y and G provided a higher level of protection against Eimeria infection compared to encapsulated phage V and the unencapsulated phages. Molecular docking studies revealed that three peptides, particularly Y and G, efficiently bind to the EtCab protein through hydrogen bonds. This study provides a reference for developing small molecular drugs targeting coccidiosis.

Introduction

Avian coccidiosis is an intestinal disease in poultry caused by one or more species of the protozoan genus Eimeria. These Eimeria parasites invade the intestinal epithelial cells, leading to acute enteritis and tissue necrosis, which manifest as clinical symptoms of diarrhoea, dehydration, and weight loss [1, 2].

Among the seven known Eimeria species, Eimeria tenella predominantly targets cecal epithelial cells, resulting in cecal hemorrhagic lesions. The life cycle of Eimeria consists of two main stages: the exogenous phase (sporogenesis) and the endogenous phases (schizogenesis and gametogenesis) [3].

During sporogenesis in the intestinal tract, sporozoites are released from sporulated oocysts. These sporozoites attach to the intestinal epithelial cells, initiating a complex invasive process in which a variety of sporozoite-secreted proteins are involved [2]. In the schizoite phase, merozoites develop from sporozoites and progress through intestinal epithelial cells, developing into large and small gametocytes. This process is followed by sexual reproduction to form zygotes, which develop into oocysts [2]. These oocysts are released from the intestinal epithelial cells and excreted with the intestinal contents. Under suitable environmental conditions, the oocysts develop into sporulated oocysts. A new cycle of sporogenesis begins when chickens orally ingest these sporulated oocysts.

The long-term use of anticoccidial drugs has led to the emergence of drug-resistant parasites, posing a significant challenge to the global poultry industry [4]. Additionally, the presence of drug residues in poultry products raises potential concerns for food safety and human health [5]. Live attenuated vaccines also present challenges for poultry health, as large-scale infections may lead to outbreaks due to the reversion of virulence in attenuated vaccines.

These drawbacks associated with traditional control methods highlight the urgent need to develop novel, cost-effective, and safe preventive measures to protect the health and well-being of chickens worldwide.

Eimeria is a site-specific intracellular parasite that requires adhesion to the surface of host cells for subsequent invasion [6]. Various proteins have been identified as important for this invasion, including microneme (MIC) proteins like E. tenella MIC3 (EtMIC3) [7] and apical membrane antigen 1 (AMA1) [8, 9]. Recent studies have shown that calcium ions (Ca2+) are essential for intercellular communication, and calcium-binding proteins (CBPs) play a significant role in regulating various cellular functions [10].

CBPs exhibit several structural characteristics, including a specific Ca2+ binding structural domain known as EF-Hand, which features a helix-loop-helix structure and is highly conserved across different CBPs [11].

Research has shown that certain CBPs located on the surface of parasites play a significant role in the adhesion and invasion of protozoan parasites. For instance, Toxoplasma gondii CDPK1 (TgCDPK1) is involved in adhesion and invasion by regulating the secretion of TgMIC2 [12], while TgCDPK4 may modulate the invasion of these parasites [13]. Similarly, Plasmodium falciparum CDPK1 (PfCDPK1) is crucial for regulating merozoite motility during egress and invasion [14]. Recently, the E. tenella Cab (EtCab) protein has been identified as essential for adherence during its invasion of host cells [15].

M13 phages were utilised to display various exogenous proteins or peptides on the surface of pIII capsid proteins; this is known as phage display technology [16]. The results showed that functional peptides screened from a phage display library, using sonicated sporozoites of E. acervulina and E. tenella as affinity ligands, can specifically bind to sporozoites. This binding effectively inhibits their invasion into host cells [17].

Additionally, a phage display library displaying single-chain variable fragments (scFv) was constructed to identify peptides with a strong ability to bind specifically to E. tenella sporozoites and merozoites [3, 18]. In the current study, we focus on the crucial role of the EtCab protein in sporozoite invasion into host cells. We hypothesise that peptides that specifically bind to the EtCab protein could effectively inhibit this invasion.

To test this hypothesis, four rounds of biopanning were conducted using twelve peptide libraries, with the EtCab protein serving as a ligand. Furthermore, since it has been reported that biocompatible material such as sodium alginate can protect encapsulated biomaterials from degradation, we examined the effect of the sodium alginate hydrogel that encapsulates bacteriophage peptides specifically binding to the EtCab protein. This effect was investigated against E. tenella sporozoite invasion both in vivo and in vitro. Additionally, a three-dimensional structural model of the EtCab protein was constructed, and molecular docking between the affinity peptide and the EtCab model was analysed.

Materials and methods

Animals and parasites

One-day-old specific pathogen-free (SPF) chickens were obtained from the Harbin Veterinary Research Institute in Heilongjiang, China, and raised in a coccidia-free environment. The E. tenella strain used in this study was maintained in our laboratory, and sporulated oocysts of E. tenella were passaged in chickens every three months. The collection of sporulated oocysts and the preparation of purified sporozoites were conducted as described previously [19].

All animal experiments were carried out at the Veterinary Hospital of Northeast Agricultural University in China. The experimental protocols adhered to the regulations set by the Ethics Committee for Animal Sciences at Northeast Agricultural University, Heilongjiang Province, China (NEAUEC20220335).

Preparation of rabbit antisera against EtCab

The rabbit antisera against the EtCab protein was prepared with some modifications based on the previously reported methods [7]. The plasmid pUC57-EtCab (preserved in our laboratory; the GenBank accession number for EtCab is XP_013229050.1) was digested using the restriction enzymes BamH I and XhoI. The resulting EtCab gene fragment was ligated into a BamH I/XhoI-digested pET-30a(+) vector. The positive plasmid pET-30a(+)-EtCab was then transformed into E. coli BL21 (DE3).

The expression of the EtCab protein in the supernatant from the ultrasonic lysate of recombinant positive bacteria was analysed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The purification of EtCab protein was conducted using nickel (Ni2+)-nitrilotriacetic (NTA) His Bind Resin (Merck, Darmstadt, Germany) following the established methods [8]. SDS-PAGE subsequently verified the purification of the target protein.

An emulsion was prepared by mixing 1.0 mg of purified EtCab protein with an equal volume of Freund’s complete adjuvant (Sigma Aldrich, StLouisMO, USA), serving as the immunogen for immunising New Zealand white rabbits (provided by the Harbin Veterinary Research Institute, Harbin, China) through subcutaneous injection. One week later, the rabbits were given another dose of recombinant EtCab protein (1.0 mg) and an equal volume of Freund’s incomplete adjuvant (Sigma Aldrich, USA). The following two immunisations were conducted at weekly intervals.

After the final immunisation, serum was collected and stored at −20 °C. The titer of rabbit polyclonal antisera against the EtCab protein was measured using an indirect enzyme-linked immunosorbent assay (ELISA), as described in a previous report [20].

Specific detection of antisera for the EtCab protein

Western blot was used to evaluate the specificity of rabbit antisera against the natural EtCab protein in E. tenella sporozoites. The methods for purifying sporozoites, as well as sonication and western blotting, were performed as previously described [21]. Additionally, an indirect immunofluorescence assay (IFA) was employed to demonstrate that the prepared antisera specifically reacted with the EtCab protein on the surface of E. tenella sporozoites.

In brief, 1.0 × 105 MDBK cells were seeded into each well of a 96-well plate. Once the cells reached 50% confluence, 3.0 × 105 sporozoites were added to each well and incubated for 4 h. Following incubation, the wells were washed three times to remove any uninvaded sporozoites. Next, 1.0 mL of 4% paraformaldehyde was added to fix the cells for 40 min. After another washing step, 1 mL of glycine (0.1M/L) was added to each well and incubated for 15 min. Then, 1 mL of Triton X-100 (0.1%) was added to each well and incubated at 37 ℃ for 15 min.

The cells were then incubated with rabbit anti-EtCab protein polyclonal sera (1:100) at 4 ℃ overnight. Following this, the cells were treated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:100) (Solarbio, Beijing, China) for 2 h. DAPI was added and allowed to incubate for 20 min. Fluorescence images were captured using the EVOS M700 inverted fluorescence microscope (Thermo, Massachusetts, USA).

Screening for phages that specifically bind to EtCab

The process for screening EtCab protein affinity phages was conducted as outlined in previous reports and followed the protocols provided in the phage display peptide library kit from New England Biolabs, USA. The size of the phage library was 1.0 × 109, and each phage displayed five identical peptides on its surface.

In brief, 100 μL EtCab protein (at a concentration of 100 μg/mL) was coated in each well of a 96-well plate. After incubating overnight at 4 ℃, the plate was blocked with 2% BSA for 2 h and then washed five times with Tris buffer containing 0.1% Tween-20 (TBST, pH 7.4). Next, 100 μL of the phage display library (at a 1:100 dilution, totalling 1.5 × 1014 pfu) was added to the wells and incubated at 25 ℃ for 30 min. The plate was then washed five times with TBST and once with 0.2 mol/L glycine–HCL (pH 2.2). After a 10-min incubation at 25 ℃, 1.0 mol/L Tris–HCL (pH 9.1) was added to neutralise the PH.

The eluent collected from the 96-well plate constituted the first round of biopanning bacteriophages. The titer of the target phage was determined and amplified in E. coli ER2738 according to the instructions provided in the phage display peptide library kit (New England Biolabs, USA). The subsequent three rounds of biopanning were conducted using the same method, with the concentration of Tween-20 in TBST increased to 0.5% and the number of washes increased to 20 times.

In the fourth round, the amplified bacteriophages were again cultured in E. coli ER2738, and after titration, 30 randomly selected bacteriophages were stored at −20 °C for more analysis.

Titre determination of target phages

Bacteriophage titre was determined using a modified double agar overlay plaque assay [22]. In brief, LB medium was utilised to dilute the phages to a suitable concentration, ranging from 108 to 1011 for amplified-PEG/NaCl concentrated phage culture supernatants and from 101 to 104 for unamplified panning eluates.

To start the assay, 10 μL of the phage was added to 200 μL of the host strain ER2738 (OD600 = 0.5) during its logarithmic phase, and the mixture was incubated with shaking at room temperature for 10 min. The infected bacteria were added to 3 mL of top agar medium, mixed immediately, and poured over preheated LB/IPTG/Xgal medium at 37 °C. After allowing the mixture to cool for 10 min, the plate was inverted and cultured at 37 °C overnight.

$${\text{The formula for calculating titer is}}:{\text{ pfu}}/{\text{mL}}\, = \,{\text{Number of plaques}}\, \times \,{1}00\, \times \,{\text{reciprocal of counted dilution}}.$$

The formula for calculating titer is: pfu/mL = Number of plaques × 100 × reciprocal of counted dilution.

The ability of selected phages to bind to the EtCab protein

The binding capability of selected phages to the EtCab protein was assessed using ELISA, following previously established protocols [7]. The plate was coated with 10 μg of EtCab protein per well and incubated overnight. After incubation, the plate was washed with PBST containing 0.5% Tween-20 and then blocked with 5% skim milk. Following this, 1.0 × 1012 pfu of the selected phages were added and incubated for 2 h.

The primary antibody, rabbit anti-M13 polyclonal antibody, was then used, diluted to 1:3000. For detection, a secondary antibody, goat anti-rabbit IgG (1:1000) conjugated with horseradish peroxidase (HRP) from Sigma (USA), was applied. Subsequently, a substrate solution containing 0.01% H2O2 and 1 mg/mL o-phenylenediamine was subsequently added for the reaction, which was then terminated using 2 M sulfuric acid. The OD450 value for each well was recorded.

For control measures, phages that bound to the N protein of infectious bronchitis virus (IBV), stored in our lab, were used as a negative control, while phages binding to the EtMIC3 protein were designed as the positive control [7]. Each phage detection assay was performed in triplicate.

Sequence analysis of target phages

DNA from selected high-affinity phages was extracted, and PCR amplification was conducted using the primer pair provided in the phage display peptide library kit from New England Biolabs, USA. After observing the target bands, the PCR product was sequenced, determining twelve peptide sequences corresponding to the target phages. The affinity peptides were then synthesised by GenScript Co., Ltd (Nanjing, China).

Binding of EtCab protein to target phages

To evaluate the binding capability of the recombinant EtCab protein and sporozoite protein to target phages, both ELISA and competitive ELISA were conducted as previously described [7].

To determine the binding capability between EtCab protein and target phages, 100 μL of affinity phages, serially diluted from 1 × 1012 to 1 × 106 pfu, was added to a 96-well plate and incubated overnight. After blocking, each well received 100 μL of purified EtCab protein at a 100 μg/mL concentration, followed by 100 μL of HRP-labeled goat anti-rabbit IgG. The OD450 value was measured in triplicate for each sample.

For the competitive ELISA, 50 μL of rabbit anti-EtCab antiserum was diluted at concentrations of 1:1000, 1:1500, 1:2000, and 1:2500, corresponding to 60, 40, 30, 24, and 20 μg/mL, respectively. This was combined with 50 μL of EtCab protein (100 μg/mL) as the binding competitor and added to each well containing 100 μL of coated positive phage (1.0 × 1012 pfu). After incubation, OD450 values were recorded, and each sample was tested in triplicate. The negative and positive phages described above were included as controls.

The binding capability of sporozoites protein to the target phages was assessed using the same method as described earlier. However, in this process, 100 μL of sonicated (300 W, 5s, 5s intervals) sporozoites (1.0 × 106/mL) protein (1.2 mg/mL) was added to interact with the coated target phages. Each sample was tested in triplicate. The negative and positive phages mentioned previously were included as controls.

Purification and labelling of sporozoites

The purification of E. tenella sporozoites was carried out using the previously reported method [7]. First, preserved sporulated E. tenella oocysts were washed and treated with a sodium hypochlorite solution. The targeted oocysts were then collected, resuspended, washed and agitated with glass beads to break the oocyst wall. The released sporocysts were collected, washed and resuspended before treatment with 0.25% trypsin and 4% taurocholic acid sodium salt to complete the excystation process. The purified sporozoites were obtained by centrifuging the samples in Percoll (55%).

Next, 5.0 × 106 sporozoites were labelled with CFDA-SE (Beyotime, China) according to the provided protocols. The labelled sporozoites were then cultured in DMEM medium (Gibco, Leicestershire, UK) at 37 °C for 30, 60, and 90 min. Labelling efficiency was assessed using a fluorescence microscope (Nikon) and flow cytometry.

Inhibition of sporozoites invasion into cells invitro

To determine the maximum non-toxic concentration of the three synthesised peptides on MDBK cells, the cells were incubated with the peptides at concentrations of 125, 250, 500, and 1000 μg/mL for 2 h at 37 °C in a humidified atmosphere containing 5% CO2. After each incubation period, the medium was discarded, and the cells were washed three times with PBS. Subsequently, serum-free DMEM (100 μL/well) and CCK-8 reagent (10 μL/well) were added for 1 h at 37 °C. The absorbance of each well was measured using an ELISA reader (BioTek, Elx808), and cell viability was calculated. Additionally, any morphological changes in the cells were observed.

The purification and labelling of sporozoites with CFDA-SE were performed using the abovementioned method. DMSO was utilised to dilute the three affinity peptides to concentrations of 125, 75, and 25 μg/mL and to dilute rabbit anti-EtCab polyclonal serum at ratios of 1:100, 1:200, and 1:300. The sporozoites were incubated with either the polyclonal serum or the affinity peptides in a cell culture plate for 1 h at 37 °C and 5% CO2, after which they were collected by centrifugation. The harvested sporozoites were then added to a monolayer of MDBK cells (1.0 × 105 per well). Following 4 h of incubation, the cells were washed three times with PBS to remove uninvaded sporozoites and then digested with trypsin (0.25%). The green fluorescence signal in the intracellular MDBK cells was detected by flow cytometry to calculate the sporozoite invasion rate. The invasion rate and inhibition rate were calculated using the following formulas:

$${\text{Invasion rate}}\, = \,\left( {\text{count of cells with green fluorescence}} \right)/({\text{count of cells with and without fluorescence}}).$$

Invasion inhibited rate = [1−(count of cells in the group treated with target peptides or anti-EtCab antisera/count of cells in the group without treatment with peptides or anti-EtCab antisera)] × 100%.

Release characteristics of target phages from microspheres in both in vivo and in vitro conditions

To prepare phage-encapsulated microspheres, 100 μL of affinity phage (1.0 × 1013 pfu) was mixed thoroughly with 10 mL of a 1.5% sodium alginate solution. This mixture was then added dropwise to a 0.1 mol/L calcium chloride solution using a syringe while stirring continuously for 1 h to allow the microspheres to harden. The encapsulation efficiency (EE) was calculated using the formula: EE = (entrapped phages/total phages) × 100%.

To test the stability of the microspheres, 100 mg of microspheres and 100 μL of free phages were combined with 900 μL of simulated gastric fluid (SGF), which consisted of 154 mM NaCl and 0.3% (w/v) pepsin at pH2.0 [23]. The mixture was gently agitated at 41 °C. Samples of the microspheres were taken at 10, 30, 60, 90, and 120 min, washed with SM buffer (0.25 g MgSO4·7H2O pH 7.5) [24], and dissolved in 1.0 mL of a microcapsule-broken solution (MBS) containing 50 mM sodium citrate tribasic dehydrate and 200 mM sodium bicarbonate in a 50 mM Tris–HCl buffer at pH 7.5 [24]. The phage titers were then measured.

The release of target phages from the microspheres in simulated intestinal fluid (SIF), which contained 0.3% (w/v) bile salt, 111 mM NaCl, 11 mM KCl, 2 mM CaCl2, 16 mM NaHCO3, and 1% (w/v) trypsin at pH 7.0 [23], was assessed as follows. 100 mg of microspheres were incubated in SGF (pH 2.0) at 41 °C for 2 h. After washing with SM buffer (pH 7.5), the microspheres were transferred to a new tube containing 900 μL SIF (pH 6.7) and incubated at 41 ℃. At intervals of 0, 5, 15, 30, 60, 90, and 120 min, 100 μL samples were taken to measure phage titers. The incubation was terminated by adding SM buffer (pH 7.5), and a phage release curve was plotted based on the phage titer and time. Each sample was tested in triplicate.

To measure the distribution of target affinity phages in the intestinal tract of chickens, sodium alginate microspheres encapsulating the target phages and free phages were orally administered to the chickens at a dose of 1.0 × 1010 pfu. At 1 and 2 h post-oral administration, contents from the gastric, duodenum, ileum, jejunum and cecal regions were collected and placed in MBS solution (pH 7.5). The phage titers were measured and curves depicting phage titers at different intestinal sites at different times were drawn. The phage titers were calculated using the formula: titers of phages = log10 (number of phage plaques × dilution ratio × total volume of sample ÷ 10). The value 10 indicates that the number of phage plaques is derived from a 10 µL diluted sample when only one phage plaque is observed on a plate made with stock solution without any dilution. The formula is as follows: log10 (1 × 1 × 2500/10) = 2.39794, representing the lower detection limit.

In vivo anticoccidial effects of target phages

At 10 days of age, the chickens were randomly divided into nine groups, with 10 chickens in each group. At 21 days of age, all groups, except for group 1 (TBS group), were orally challenged with 1.0 × 104 E. tenella sporulated oocysts. On days 0, 1, and 2 post-challenge, the chickens in group 2 (phage Y + Alg), 3 (phage G + Alg), and 4 (phage V + Alg) were orally gavaged with 1.0 × 1010 pfu of phage Y, G, and V encapsulated in sodium alginate microspheres, respectively. Chickens in groups 5 (phage Y), 6 (phage G), and 7 (phage V) received 1 × 1010 pfu of free phage Y, G, and V without encapsulation. Group 8 received sodium alginate microspheres (Alg), while group 9, the E. tenella challenge control, was given TBS (pH7.2).

The chickens in each group were weighed both before the E. tenella challenge and on day 7 post-challenge to calculate body weight gain (BWG). Faecal samples were collected from individual chickens in each group between days 5 and 7 post-challenge to calculate oocysts per gram (OPG). Additionally, cecal lesion scores and oocyst reduction rates were recorded as previously described [19].

All animal experiments followed the regulations set by the Ethics Committee for Animal Sciences at Northeast Agricultural University, Heilongjiang Province, China (NEAUEC20220335).

Histopathological examination in cecal tissues

Cecal tissues from chickens in each group were collected, rinsed gently with PBS, and stored in 10% formalin for histopathological examination. After 1 week of fixation, the tissue specimens were trimmed, rinsed with tap water overnight to remove the fixative, and then dehydrated using alcohol of different concentrations. Subsequently, the tissues were clarified in xylene and embedded in paraffin. The paraffin-embedded block was cut into 4 μm thick sections, stained with hematoxylin and eosin (H&E staining) [25], and finally mounted with neutral resin for observation and photography.

Homologous modelling, molecular docking, and homology analysis

The protein-coding gene sequence for EtCab was obtained from the UniProt database. Protein secondary structure analysis was conducted using the online SOPMA software [26]. The 3D structure of the target sequence was predicted using the I-Tasser server, which recognised the template sequence through Lomets [27]. This server was also applied to optimise the models [28]. Online software was utilised to evaluate the stereochemical characteristics of the constructed models [29].

Homologous modelling of the three target peptides was performed using Modeller 10.1 software, further optimised through the Chiron portal. The ClusPro [30] website was used for the flexible docking of proteins and target peptides, while pymol was employed for drawing and analysis. Sequence comparison was conducted using ESPript3.0 [31].

Sequences used for analysing membrane-associated calcium-binding proteins from E. necatrix (CDJ63198.1), E. acervulina (XP_013252173.1), E. mitis (XP_013357999.1), and E. maxima (XP_013337521.1) were retrieved from GenBank.

Statistics and analysis

Statistical analysis was conducted using SPSS software v26.0 (SPSS Inc., Chicago, IL, USA). The analysis involved a one-way analysis of variance (ANOVA) along with Duncan’s multiple-comparison procedures. Data were presented as mean ± SD. A p-value of less than 0.05 was considered statistically significant, while a p-value of less than 0.01 was deemed highly significant.

Results

Characterisation of polyclonal antisera against EtCab protein

The titre of the prepared polyclonal antisera against the EtCab protein was 1: 216 (Figure 1A). Western blot analysis demonstrated that the antiserum specifically reacted with E. tenella sporozoites, producing a single protein band at 66 kDa (Figure 1B). Indirect immunofluorescence assays indicated that the antisera against the EtCab protein effectively reacted with E. tenella sporozoites as they invaded MDBK cells, confirmed by a distinct green fluorescence signal (Figure 1C).

Figure 1
figure 1

Detection of prepared polyclonal antisera against EtCab protein. The titre of the prepared antisera against the EtCab protein was 1:216. The positive cutoff = 2 × OD450 value of negative control (A). Western blot analysis was performed to detect the specific reaction of the anti-EtCab antisera with E. tenella sporozolites, revealing a band of 66 kDa (B). An indirect immunofluorescence assay (IFA) showed that the anti-EtCab antisera specifically reacted with the EtCab protein of E. tenella sporozoites, producing a green fluorescence signal (C). The titre of each sample was tested in triplicate, and the values are presented as mean ± SD (n = 5).

Full size image

Screening of phages that bind to EtCab and conducting binding assays

The plaque-forming units per millilitre (pfu/mL) of the positive phages binding to the EtCab protein were identified following four rounds of affinity screening (Table 1), indicating that the enriched phages could bind to the EtCab protein. The results from the ELISA demonstrated that the binding ability of all thirty selected bacteriophages to the EtCab protein was significantly higher than that of the blank control (BC) and the negative bacteriophage control (NC) (P < 0.001) (Figure 2A).

Table 1 Titre determination of phage elution (pfu)
Full size table
Figure 2
figure 2

Affinity determination of screening bacteriophage with EtCab protein. The binding capability of thirty randomly selected phages was found to be higher than that of the blank control (BC) and the negative control (NC). The positive control (PC) phages, designed to bind to the EtMIC3 protein, were prepared as shown in (A). Phages were diluted from 1.0 × 1012 to 1.0 × 106 pfu/10 μL and coated in each well of a 96-well plate. The binding abilities of phages Y, G, and V were greater than that of the NC phages, with phages Y and G exhibiting higher binding than phage V (B). When 1.0 × 1012 pfu/10 μL of phages Y, G, and V were coated per well in a 96-well plate, their binding abilities to the recombinant EtCab protein and sporozoites lysates supposed those of the NC phages (C). The binding of the three phages to the EtCab protein was significantly inhibited by anti-EtCab antisera when compared to an irrelevant polyclonal antibody (PcAb) against the ΔHexon protein of fowl adenovirus serotype 4 (FAdV-4), which served as the negative antibody control (D). Each sample was tested in triplicate and the values represent the mean ± SD (n = 5). Statistical significance is denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and “ns” indicates no statistical difference.

Full size image

The sequences of twelve peptides from the thirty corresponding bacteriophages are summarised in Table 2. Among these, three sequences—YLNPINHRLTNP (Y), GDRMNVIWDTFA (G) and VSNGFHLHLELR (V)—exhibited the highest repetition ratios and were selected for subsequent experiments.

Table 2 The deduced amino acid sequences of the selected thirty positive phages clones
Full size table

All three target phages Y, G, and V were able to bind to the EtCab protein. However, the affinity of target phages Y and G for the EtCab protein was significantly greater than that of phage V (P < 0.001) (Figure 2B). The binding ability of bacteriophages Y, G, and V to recombinant EtCab protein and sporozoite ultrasonic lysates was significantly higher than that of unrelated bacteriophages (selected from the N protein of the infectious bronchitis virus) (P < 0.001) (Figure 2C).

Competitive ELISA results demonstrated that as the concentration of EtCab antisera increased, the antibodies competed with the three affinity bacteriophages to bind to the EtCab protein. In contrast, the unrelated rabbit polyclonal antibody against avian adenovirus serotype 4 (FADV-4) Hexon protein did not show dose-dependent inhibition (Figure 2D).

Inhibition of sporozoites invasion into cells in vitro

Fluorescence microscopy revealed that 97.4% of sporozoites were labelled with a fluorescent signal using the CFDA-SE kit. These labelled sporozoites could be detected in MDBK cells just 2 h after incubation, indicating their ability to invade MDBK cells (Figure 3A). However, the longer the sporozoites were incubated in vitro, the lower their invasion rate into MDBK cells became (Figure 3B).

Figure 3
figure 3

In vitro model of E. tenella sporozoites invasion into MDBK cells. Approximately 97.4% of freshly purified E. tenella sporozoites were labelled using CFDA-SE (A). The labelled sporozoites were incubated in DMEM medium at 37 °C for time periods of 30, 60, and 90 min, before being co-incubated with MDBK cells for 2 h. Flow cytometry results indicated that as the duration of in vitro incubation of the sporozoites increased, the efficiency of their invasion into the cells decreased (B). Each sample was tested in triplicate, and the values are presented as mean ± SD (n = 3). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and “ns” indicates no statistical difference.

Full size image

The maximum non-toxic concentration of the synthesised affinity peptides Y, G, and V for MDBK cells was 125 μg/mL (Figure 4A). Compared to negative sera, rabbit anti-EtCab polyclonal antisera at various dilution ratios inhibited the invasion of E. tenella sporozoites into MDBK cells. Notably, the inhibitory effects diminished as the dilution ratio increased (Figure 4B).

Figure 4
figure 4

Inhibition of sporozoites invasion into MDBK cells by target peptides in vitro. The maximum non-toxic concentration of the three peptides was found to be 125 µg/mL (A). The labelled sporozoites were incubated with anti-EtCab antisera diluted 1:100, 1:200, and 1:300, respectively, before being added to MDBK cells. The dilution of anti-EtCab antisera at 1:100 resulted in the lowest invasion rate and the highest rate of invasion inhibition (B). Additionally, the labelled sporozoites were incubated with concentrations of 125, 75, and 25 μg/mL of the three peptides, and then introduced to MDBK cells. At a concentration of 125 μg/mL, peptides Y and G exhibited a higher rate of invasion inhibition (C). Each sample was tested in triplicate, and the values are expressed as mean ± SD (n = 3). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and “ns” indicates no statistical difference.

Full size image

All three affinity peptides demonstrated a significant ability to inhibit sporozoite invasion into MDBK cells in a dose-dependent manner. At every concentration tested, the inhibitory effects of peptides Y and G were significantly greater than that of V peptide (P < 0.001) (Figure 4C).

Release and distribution of encapsulated target phages

The encapsulation efficiency was 97.47 ± 4.62%. Free phages that were not encapsulated completely lost their activity after incubating in simulated gastric juice (pH 2.0) for 30 min. In contrast, the titre of phages coated with sodium alginate (Alg-coated) only decreased by 1.62 ± 0.24 log10 pfu/g after 120 min of incubation (Figure 5A). This indicates that the phages encapsulated in sodium alginate microspheres were protected from degradation in simulated gastric juice (pH 2.0).

Figure 5
figure 5

Release and distribution of target phages encapsulated by sodium alginate. Phages, both encapsulated and with sodium alginate (Alg-coated) and free (without encapsulation), were incubated in simulated gastric fluid (SGF) at pH 2. The free phages completely lost their activity after 30 min of incubation, while the titres of encapsulated phages decreased by only 1.62log10 pfu/g after 120 min (A). After being incubated in SGF for 2 h, the encapsulated phages transferred to simulated intestinal fluid (SIF) at pH 6.7 for further incubation. During this time, the titres of phages in the supernatant gradually increased. At 30 min, 32.9% of the encapsulated phages had been released, and at 120 min, 92.6% were released (B). 1 h after oral administration, the titres of phages in the duodenum, jejunum, and ileum were higher than those in the gizzard, cecum, and rectum. However, at 2 h post administration, the titres in the cecum were slightly higher than in the duodenum and gizzard (C and D). Values are expressed as mean ± SD (n = 6). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, and “ns” indicates no statistical difference.

Full size image

When the Alg-coated microspheres were transferred from simulated gastric juice (pH 2.0) to simulated intestinal fluid (pH6.8), the titre of phages in the supernatant gradually increased. At 30 min, 32.9% of the encapsulated phages were released, and at 120 min, 92.6% were released (Figure 5B).

Following oral administration, the distribution of the target phage Y in the chicken digestive tract was evaluated, as shown in Figure 5C and D. The titre of target phages in the gizzard for the Alg-coated group was significantly higher than that of the free phage group at 1 h (P < 0.01), indicating that some unencapsulated phages were degraded after oral administration. Although the titres of free phages in the jejunum and ileum were significantly higher than those in the Alg-coated group (P < 0.01) at 1 h, the titre of phages in the jejunum in Alg-coated group at 2 h was significantly higher than that in the free phage group (P < 0.05). This suggests that phages encapsulated by sodium alginate could be gradually released and remain at high titres in the anterior digestive tract for a relatively long time.

Evaluation of anti-coccidial effects in vivo

As shown in Table 3, the average body weight gain (WG) in the groups that received oral administration of encapsulated and unencapsulated bacteriophages was significantly higher compared to the infection control group (P < 0.05). The WG in the sodium alginate encapsulation group was considerably higher than in the unencapsulated group (P < 0.05). Furthermore, the WG in the phage Y and G groups was significantly higher than that in the phage V group (P < 0.05).

Table 3 Changes of weight gain of chickens in each group
Full size table

The oocyst decrease ratio in the phageY + Alg, phageG + Alg, phageV + Alg, phageY and phageG groups was greater than that in the phageV, Alg, and infection control groups (Figure 6A). The cecal lesion scores in the phageY + Alg, phageG + Alg groups were lower than those in the phageY, phageG, phageV + Alg and phage V groups, which were also lower than in the infection and Alg control groups (Figure 6B).

Figure 6
figure 6

Lesion score and oocyst decrease ratio. 7 d after the challenge with E. tenella, the groups treated with phageY + Alg, phageG + Alg, phageV + Alg, phageY, and phageG showed a greater decrease in oocyst counts compared to those treated with phageV, Alg, and the infection control group (A). The caecal lesion scores for the phageY + Alg and phageG + Alg groups were lower than those for the phageY, phageG, phageV + Alg, and phage V groups, all of which has lower scores than the infection and Alg control groups (B). Values are expressed as mean ± SD (n = 10). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01.

Full size image

To illustrate the protective effects, gross and histopathological changes in the ceca were documented. In the TBS group (Figure 7A), the ceca of chickens appeared normal, with no pathological changes observed. In contrast, the ceca in the E. tenella infection control and Alg control groups (Figures 7H and I) were small, exhibiting thickened caecal walls, scattered haemorrhage spots, and grey lesions protruding from the serous surface. Only slight caecal swelling was noted in the phageY + Alg, phageG + Alg, and phageY groups (Figures 7B, C and E). Additionally, slight cecal swelling and a few lesions on the serosal surface were observed in the phageG group (Figure 7F). In contrast, cecal swelling with serous haemorrhagic spots was observed in the phageV + Alg and phageV groups (Figures 7D and G).

Figure 7
figure 7

Gross pathological lesions in ceca of chickens in each group. 7 days after the challenge with E. tenella, no obvious lesions were observed in the ceca of chickens in the TBS control group (A). In the phageY + Alg (B), phageG + Alg (C), and phageY (E) groups, slight swelling of the ceca was noted. The phageG group exhibited slight caecal swelling along with a few lesions on the serosal surface (F). More pronounced caecal swelling with serous haemorrhagic spots was observed in both ther phageV + Alg and phageV groups (D and G). In the E. tenella infection group (H) and the Alg group (I), the cecal wall appeared thickened, with scattered haemorrhagic spots and grey lesions on the serosal surface.

Full size image

Figure 8 demonstrates that no significant histopathological changes were observed in the cecal tissues of the TBS group. In the E. tenella infection and Alg control groups, partial intestinal villi were damaged, epithelial cells had shed into the enteric cavity, inflammatory cell infiltration was noted in the lamina propria, oedema in the submucosa, and the muscle layer was visibly thickened. Notably, intestinal gland epithelial cells in the lamina propria were invaded by large numbers of E. tenella at various developmental stages, including macrogametes, microgametes, and oocysts, which resulted in severe structural damage. In the phageY + Alg group, the structure of the intestinal villi remained normal, and the quantity of E. tenella parasites was significantly lower than that in the infection control group. In the phageG + Alg, phageY, and phageG groups, the structure of the intestinal villi was partially broken, with infiltrated inflammatory cells present, but the number of E. tenella was not as severe as in the E. tenella infection and Alg control group. The histopathological changes in the phageV + Alg and phageV groups were similar to those observed in the E. tenella infection control group (Figure 8).

Figure 8
figure 8

Histopathological changes in ceca tissues from chickens in each group. No obvious histopathological changes were observed in the ceca tissues of the TBS group. In the infection and Alg control groups, some intestinal villi were damaged, featuring infiltration of inflammatory cells in the lamina propria, oedema in the submucosa, and a marked thickening of the muscle layer. Intestinal gland epithelial cells in the lamina propria were invaded by a substantial number of Eimeria parasites at various developmental stages, including macrogametes, microgametes, and oocysts. In the phageY + Alg group, the structure of the intestinal villi appeared relatively normal, and the number of parasites was lower than in the E. tenella infection group. In the phageG + Alg, phageY, and phageG groups, the structure of the intestinal villi was partially damaged but the infiltration of inflammatory cells and the number of parasites were less compared to the E. tenella infection and Alg control groups. In both the phageV + Alg and phageV groups, the histopathological changes were similar to those observed in the E. tenella infection group.

Full size image

Analysis of the binding interactions between target peptides and the EtCab protein through in silico methods

The ProtParam web server analysis revealed that the EtCab protein consists of 304 amino acid residues. A secondary structure analysis conducted using SOPMA indicated that the EtCab protein contained four structural forms: 58.22% α-helix, 5.59% extension chain, 8.55% β-rotation and 27.63% random crimp (Additional file 1). The 3D structure of the EtCab sequence was predicted using the I-Tasser server, along with the C-scores of the four top-ranking models (Figure 9A). The 3D model with the highest score was optimised, and its stereochemical properties were evaluated using a Ramachandran Plot (Additional file 2).

Figure 9
figure 9

Prediction of 3D model for EtCab protein and molecular docking with target peptides. Molecular docking analysis was conducted to evaluate the binding of Y (B), G (C), and V (D) peptides to the EtCab protein. The EtCab protein is shown in green, while the polypeptides are highlighted in red. Dotted lines indicate hydrogen bonds, and the numbers reflect bond lengths (with shorter bond lengths correlating to greater bond energy). The label ASN-3 specifies that position 3 contains an ASN amino acid. The I-Tasser server generated the top four models and their corresponding ratings (A). The sequence alignment of Cab protein amino acids from five Eimeria species is displayed. Amino acids with a red shadow are completely conserved, those in red font red are partially conserved, amino acids bound to the short peptide are highlighted in green, and amino acids that interact with the Y and G peptides are indicated in blue (E).

Full size image

Molecular docking results demonstrated that all three affinity peptides corresponding to the three phages could bind to the EtCab protein through hydrogen bonding. The binding strength was highest for the Y peptide(B) at −874.6, followed by the G peptide(C) at −628.8, and the V peptide(D) at −511.3. The amino acids involved in hydrogen bonding between the three peptides and the EtCab are listed in Table 4.

Table 4 Amino acids that predicted for forming hydrogen-bonds between EtCab protein and peptide Y, G and V
Full size table

To further investigate the conserved sites in the EtCab protein that can be recognised by the three affinity peptides, a comparison was made of the Cab protein sequences from five Eimeria species: E. tenella, E. necatrix, E. acervulina, E. mitis and E. maxima, using DNAstar software. This analysis revealed that two conserved amino acids, Lys174 and Asp175, are common binding sites for both the Y and G peptides across the five Eimeria species (Figure 9E).

Discussion

The invasion of Eimeria parasites begins with the attachment of asexual sporozoites ito host cells. Research has shown that apical membrane antigen 1 (AMA1) [32] and microneme protein 3 (MIC3) [33] both play crucial roles in this attachment process. A recent study revealed the significant involvement of calcium-binding (Cab) proteins during the invasion of apicomplexans parasites. For example, yeast expressing the E. tenella Cab (EtCab) demonstrated a 2.5-fold higher attachment rate to host cells compared to control yeast [15], highlighting the critical role of the EtCab protein in host cell attachment.

In this study, an indirect immunofluorescence assay (IFA) showed that rabbit antisera prepared against the EtCab protein reacted with the proteins of sporozoites. This indicates that the antisera can bind to the natural EtCab protein, consistent with previous reports [15]. Furthermore, western blot analysis confirmed that the anti-EtCab antisera specifically recognised the EtCab protein, as evidenced by a distinct band. However, the size of this band was larger than anticipated (about double the predicted size), suggesting the presence of post-translational modifications, such as glycosylation or phosphorylation [34], and the formation of EtCab protein homodimers.

Phage display technology has been extensively used to discover peptides or antibodies specific to various pathogens or diseases, including bacteria [35,36,37,38], viruses [39,40,41], cancer [42, 43], and parasites [44,45,46]. A prior study demonstrated the specific binding of affinity peptides from E. acervulina and E. tenella to sporozoites [17]. In our laboratory, we utilised the adhesion regions (MARR) of E. tenella microneme protein 3 (EtMIC3) [7] and EtAMA1 proteins [8] as ligands for screening affinity peptides. The screened peptides exhibited a certain degree of anticoccidial effects.

In the present study, three identified EtCab protein-affinity peptides showed strong binding capabilities and competed with rabbit anti-EtCab antisera for binding to sporozoites protein and EtCab protein. This suggests that the binding sites of these three target peptides overlapped with those sites recognised by the rabbit anti-EtCab antisera.

Several studies have demonstrated that antibodies targeting essential proteins of E. tenella effectively inhibit the invasion of sporozoites into host cells in vitro [47,48,49,50]. In this study, we aimed to explore whether three identified affinity peptides could inhibit the invasion of E. tenella sporozoites into host cells as effectively as rabbit anti-EtCab antisera. Our findings revealed that rabbit antisera to EtCab protein, diluted at a ratio of 1:100, showed similar inhibition rates to the Y and G peptides (125 μg/mL) against sporozoites invasion into MDBK cells. This observation is consistent with competitive ELISA tests, suggesting that the affinity peptides may mimic epitopes from the anti-EtCab antisera that bind to EtCab proteins.

It is widely accepted that encapsulation in sodium alginate, a biocompatible and cost-effective material, can preserve phage viability after oral administration [51]. The pKa values of β-D-mannuronic acid and α-L-guluronic acid in the glycosyl chain of sodium alginate are higher than the pH value of simulated gastric juice, which facilitates the formation of hydrogen bonds. This results in a compact spherical structure that protects encapsulated phages from degradation. Conversely, in simulated intestinal juice, the pKa values are lower than thepH, causing the sodium alginate gel to swell. Phosphate and potassium ions then displace Ca2+ in the microspheres, gradually releasing encapsulated phages. This release process lasts approximately 1 h, aligning with the intestinal emptying time of chickens, which have short intestinal tracts.

To ensure that phages are sufficiently available at target digestive sites to efficiently bind to E. tenella sporozoites, we investigated the temporal and spatial distribution in the gastrointestinal tract following oral administration of encapsulated phages. The results showed that 1 h after oral administration, phage titres in the duodenum, jejunum, and ileum were higher than in the gizzard and posterior digestive tract (cecum and rectum). Within 2 h, phage titres in the cecum were slightly higher than those in the anterior duodenum and gizzard. A previous report indicated that 1 h after the infection with E. tenella sporulated oocysts, large numbers of free sporozoites were found in the duodenum, and 2 h later, sporozoites reached and began to invade caecal epithelial cells [2]. This suggests that the target phages have ample opportunity to contact and bind to sporozoites released in the digestive tract. We observed that orally administered phages remained detectable in the intestines 2 h post-administration, likely due to the chyme shielding the phages from digestive enzymes.

To determine whether the target phages exhibited anticoccidial effects in vivo, three different phage preparations (encapsulated and unencapsulated) were orally gavaged on days 0, 1, and 2 following oral challenge with E. tenella sporulated oocysts. The results indicated that the encapsulated phages were more effective than the unencapsulated ones in reducing the infection.

To clarify the mechanisms by which three target phages operate, we conducted molecular docking analysis. The analysis revealed that the Asn, Ile, and Arg residues in the three peptides associated with these three phages, particularly the Y and G peptides, can form hydrogen bonds with the EtCab protein. The number and strength of these hydrogen bonds correlate with the experimental results observed in vitro and the anticoccidial effects noted in vivo.

Additionally, a sequence comparison and analysis of the Cab protein from five Eimeria parasites indicated that the amino acids Lys174 and Asp175 in the EtCab protein interact not only with the Y peptide but also with the G peptide. This interaction explains the superior effects of the Y and G peptides observed in previous experiments.

Furthermore, we found that some of the amino acid sites in the Cab protein that bind to the three peptides are conserved across different species of Eimeria. This suggests that the identified affinity phages and their corresponding peptides may have broader therapeutic potential. Overall, our research provides valuable insights for developing small molecular drugs against avian coccidiosis.

Data availability

Not applicable.

Abbreviations

EtCab:

E. tenella Calcium binding

NTA:

nickel (Ni2+)-nitrilotriacetic

FITC:

fluorescein isothiocyanate

SDS-PAGE:

sodium dodecyl sulphate–polyacrylamide gel electrophoresis

IFA:

indirect immunofluorescence assay

ELISA:

enzyme-linked immunosorbent assay

HRP:

horseradish peroxidase

IPTG:

isopropyl-b-D-thiogalactopyranoside

SPF:

specific pathogen-free

3D:

three-dimensional

BWG:

body weight gain

References

  1. Chapman HD (2014) Milestones in avian coccidiosis research: a review. Poult Sci 93:501–511

    Article  CAS  PubMed  Google Scholar 

  2. Mesa-Pineda C, Navarro-Ruíz JL, López-Osorio S, Chaparro-Gutiérrez JJ, Gómez-Osorio LM (2021) Chicken coccidiosis: from the parasite lifecycle to control of the disease. Front Vet Scie 8:787653

    Article  Google Scholar 

  3. Abi-Ghanem D, Waghela SD, Caldwell DJ, Danforth HD, Berghman LR (2008) Phage display selection and characterization of single-chain recombinant antibodies against Eimeria tenella sporozoites. Vet Immunol Immunopathol 121:58–67

    Article  CAS  PubMed  Google Scholar 

  4. Peek HW, Landman WJ (2011) Coccidiosis in poultry: anticoccidial products, vaccines and other prevention strategies. Vete Q 31:143–161

    Article  CAS  Google Scholar 

  5. Bafundo KW, Cervantes HM, Mathis GF (2008) Sensitivity of Eimeria field isolates in the United States: responses of nicarbazin-containing anticoccidials. Poul Sci 87:1760–1767

    Article  CAS  Google Scholar 

  6. Sibley LD (2010) How apicomplexan parasites move in and out of cells. Curr Opin Biotechnol 21:592–598

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chen W, Ma C, Li G, Jia Z, Yang X, Pan X, Ma D (2021) Specific EtMIC3-binding peptides inhibit Eimeria tenella sporozoites entry into host cells. Vet Res 52:24

    Article  PubMed  PubMed Central  Google Scholar 

  8. Ma D, Huang Y, Ma C, Zhang L, Wang J, Wang D, Li J, Dalloul RA (2019) Eimeria tenella: specific EtAMA1-binding peptides inhibit sporozoite entry into host cells. Poul Sci 98:4480–4491

    Article  CAS  Google Scholar 

  9. Péroval M, Péry P, Labbé M (2006) The heat shock protein 90 of Eimeria tenella is essential for invasion of host cell and schizont growth. Int J Parasitol 36:1205–1215

    Article  PubMed  Google Scholar 

  10. Yáñez M, Gil-Longo J, Campos-Toimil M (2012) Calcium binding proteins. Adv Exp Med Biol 740:461–482

    Article  PubMed  Google Scholar 

  11. Moreno SN, Ayong L, Pace DA (2011) Calcium storage and function in apicomplexan parasites. Essays Biochem 51:97–110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Katris NJ, Ke H, McFadden GI, van Dooren GG, Waller RF (2019) Calcium negatively regulates secretion from dense granules in Toxoplasma gondii. Cell Microbiol 21:e13011

    Article  PubMed  PubMed Central  Google Scholar 

  13. Long S, Wang Q, Sibley LD (2016) Analysis of noncanonical calcium-dependent protein kinases in Toxoplasma gondii by targeted gene deletion using CRISPR/Cas9. Infect Immun 84:1262–1273

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kato N, Sakata T, Breton G, Le Roch KG, Nagle A, Andersen C, Bursulaya B, Henson K, Johnson J, Kumar KA, Marr F, Mason D, McNamara C, Plouffe D, Ramachandran V, Spooner M, Tuntland T, Zhou Y, Peters EC, Chatterjee A, Schultz PG, Ward GE, Gray N, Harper J, Winzeler EA (2008) Gene expression signatures and small-molecule compounds link a protein kinase to Plasmodium falciparum motility. Nat Chem Biol 4:347–356

    Article  CAS  PubMed  Google Scholar 

  15. Wang Y, Zhou X, Wang H, Sun L, Wang B, Jiang Y, Li H, Zhang X, Li H, Zhao X (2021) The role of Eimeria tenella EtCab protein in the attachment and invasion of host cells. Vet Parasitol 292:109415

    Article  CAS  PubMed  Google Scholar 

  16. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317

    Article  CAS  PubMed  Google Scholar 

  17. da Silva JA, Kawazoe U, Freitas FF, Gatti MS, Dolder H, Schumacher RI, Juliano MA, da Silva MJ, Leite A (2002) Avian anticoccidial activity of a novel membrane-interactive peptide selected from phage display libraries. Mol Biochem Parasitol 120:53–60

    Article  PubMed  Google Scholar 

  18. Zhao Y, Amer S, Wang J, Wang C, Gao Y, Kang G, Bao Y, He H, Qin J (2010) Construction, screening and identification of a phage display antibody library against the Eimeria acervulina merozoite. Biochem Biophys Res Commun 393:703–707

    Article  CAS  PubMed  Google Scholar 

  19. Ma D, Ma C, Pan L, Li G, Yang J, Hong J, Cai H, Ren X (2011) Vaccination of chickens with DNA vaccine encoding Eimeria acervulina 3–1E and chicken IL-15 offers protection against homologous challenge. Exp Parasitol 127:208–214

    Article  CAS  PubMed  Google Scholar 

  20. Jia Z, Pan X, Zhi W, Chen H, Bai B, Ma C, Ma D (2022) Probiotics surface-delivering Fiber2 protein of fowl adenovirus 4 stimulate protective immunity against hepatitis-hydropericardium syndrome in chickens. Front Immunol 13:919100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ma C, Li G, Chen W, Jia Z, Yang X, Pan X, Ma D (2021) Eimeria tenella: IMP1 protein delivered by Lactococcus lactis induces immune responses against homologous challenge in chickens. Vet Parasitol 289:109320

    Article  CAS  PubMed  Google Scholar 

  22. Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP (2009) Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol Biol 501:69–76

    Article  CAS  PubMed  Google Scholar 

  23. Fayed B, Abood A, El-Sayed HS, Hashem AM, Mehanna NSH (2018) A synbiotic multiparticulate microcapsule for enhancing inulin intestinal release and Bifidobacterium gastro-intestinal survivability. Carbohydr Polym 193:137–143

    Article  CAS  PubMed  Google Scholar 

  24. Islam GS, Wang Q, Sabour PM (2018) Encapsulation strategies of bacteriophage (Felix O1) for oral therapeutic application. Methods Mol Biol 1681:71–87

    Article  CAS  PubMed  Google Scholar 

  25. Hassan SMH, Zayeda R, Elakany H, Badr S, Abou-Rawash A, Abd-Ellatieff H (2023) Anticoccidial activity of Aloe vera leafs’ aqueous extract and vaccination against Eimeria tenella: pathological study in broilers. Vet Res Commun 48:403–416

    Article  PubMed  PubMed Central  Google Scholar 

  26. Combet C, Blanchet C, Geourjon C, Deléage G (2000) NPS@: network protein sequence analysis. Trends Biochem Sci 25:147–150

    Article  CAS  PubMed  Google Scholar 

  27. Zhou X, Zheng W, Li Y, Pearce R, Zhang C, Bell EW, Zhang G, Zhang Y (2022) I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction. Nat Protoc 17:2326–2353

    Article  CAS  PubMed  Google Scholar 

  28. Ramachandran S, Kota P, Ding F, Dokholyan NV (2011) Automated minimization of steric clashes in protein structures. Proteins 79:261–270

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Colovos C, Yeates TO (1993) Verification of protein structures: patterns of nonbonded atomic interactions. Protein sci 2:1511–1519

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kozakov D, Hall DR, Xia B, Porter KA, Padhorny D, Yueh C, Beglov D, Vajda S (2017) The ClusPro web server for protein–protein docking. Nat Protoc 12:255–278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42:W320-324

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Boucher LE, Bosch J (2015) The apicomplexan glideosome and adhesins-structures and function. J Struct Biol 190:93–114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Lai L, Bumstead J, Liu Y, Garnett J, Campanero-Rhodes MA, Blake DP, Palma AS, Chai W, Ferguson DJ, Simpson P, Feizi T, Tomley FM, Matthews S (2011) The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella. PLoS Pathog 7:e1002296

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bastos LM, Macêdo AG Jr, Silva MV, Santiago FM, Ramos EL, Santos FA, Pirovani CP, Goulart LR, Mineo TW, Mineo JR (2016) Toxoplasma gondii-derived synthetic peptides containing B- and T-cell epitopes from GRA2 protein are able to enhance mice survival in a model of experimental toxoplasmosis. Front Cell Infect Microbiol 6:59

    Article  PubMed  PubMed Central  Google Scholar 

  35. Hagens S, Bläsi U (2003) Genetically modified filamentous phage as bactericidal agents: a pilot study. Lett Appl Microbiol 37:318–323

    Article  CAS  PubMed  Google Scholar 

  36. Lu TK, Collins JJ (2009) Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy. Proc Natl Acad Sci USA 106:4629–4634

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. May T, Tsuruta K, Okabe S (2011) Exposure of conjugative plasmid carrying Escherichia coli biofilms to male-specific bacteriophages. ISME J 5:771–775

    Article  CAS  PubMed  Google Scholar 

  38. Vaks L, Benhar I (2011) In vivo characteristics of targeted drug-carrying filamentous bacteriophage nanomedicines. J Nanobiotechnology 9:58

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zou N, Xia J, Wang F, Duan Z, Miao D, Yan Q, Cao S, Wen X, Liu P, Huang Y (2015) Two novel neutralizing antigenic epitopes of the s1 subunit protein of a QX-like avian infectious bronchitis virus strain Sczy3 as revealed using a phage display peptide library. Vet Immunol Immunopathol 168:49–55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zou H, Zarlenga DS, Sestak K, Suo S, Ren X (2013) Transmissible gastroenteritis virus: identification of M protein-binding peptide ligands with antiviral and diagnostic potential. Antiviral Res 99:383–390

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhu C, Zheng F, Sun T, Duan Y, Cao J, Feng H, Shang L, Zhu Y, Liu H (2013) Interaction of avian influenza virus NS1 protein and nucleolar and coiled-body phosphoprotein 1. Virus Genes 46:287–292

    Article  CAS  PubMed  Google Scholar 

  42. Romanov VI, Durand DB, Petrenko VA (2001) Phage display selection of peptides that affect prostate carcinoma cells attachment and invasion. Prostate 47:239–251

    Article  CAS  PubMed  Google Scholar 

  43. Ghosh D, Kohli AG, Moser F, Endy D, Belcher AM (2012) Refactored M13 bacteriophage as a platform for tumor cell imaging and drug delivery. ACS Synth Biol 1:576–582

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Beghetto E, Spadoni A, Buffolano W, Del Pezzo M, Minenkova O, Pavoni E, Pucci A, Cortese R, Felici F, Gargano N (2003) Molecular dissection of the human B-cell response against Toxoplasma gondii infection by lambda display of cDNA libraries. Int J Parasitol 33:163–173

    Article  CAS  PubMed  Google Scholar 

  45. Di Cristina M, Del Porto P, Buffolano W, Beghetto E, Spadoni A, Guglietta S, Piccolella E, Felici F, Gargano N (2004) The Toxoplasma gondii bradyzoite antigens BAG1 and MAG1 induce early humoral and cell-mediated immune responses upon human infection. Microbes Infect 6:164–171

    Article  PubMed  Google Scholar 

  46. Beghetto E, Pucci A, Minenkova O, Spadoni A, Bruno L, Buffolano W, Soldati D, Felici F, Gargano N (2001) Identification of a human immunodominant B-cell epitope within the GRA1 antigen of Toxoplasma gondii by phage display of cDNA libraries. Int J Parasitol 31:1659–1668

    Article  CAS  PubMed  Google Scholar 

  47. Lamarque M, Besteiro S, Papoin J, Roques M, Vulliez-Le Normand B, Morlon-Guyot J, Dubremetz JF, Fauquenoy S, Tomavo S, Faber BW, Kocken CH, Thomas AW, Boulanger MJ, Bentley GA, Lebrun M (2011) The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites. PLoS Pathog 7:e1001276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Wang B, Zhao N, Sun J, Sun L, Li H, Wu Z, Li H, Zhang X, Zhao X (2022) Identification and characterization of Eimeria tenella Rhoptry Protein 35 (EtROP35). Vet Sci 9:465

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Li H, Sun L, Jiang Y, Wang B, Wu Z, Sun J, Zhang X, Li H, Zhao X (2022) Identification and characterization of Eimeria tenella EtTrx1 protein. Vet Parasitol 310:109785

    Article  CAS  PubMed  Google Scholar 

  50. Wang Q, Zhu S, Zhao Q, Huang B, Yu S, Yu Y, Liang S, Wang H, Zhao H, Han H, Dong H (2021) Identification and characterization of a novel apical membrane antigen 3 in Eimeria tenella. J Eukaryot Microbiol 68:e12836

    Article  CAS  PubMed  Google Scholar 

  51. Malik DJ, Sokolov IJ, Vinner GK, Mancuso F, Cinquerrui S, Vladisavljevic GT, Clokie MRJ, Garton NJ, Stapley AGF, Kirpichnikova A (2017) Formulation, stabilisation and encapsulation of bacteriophage for phage therapy. Adv Colloid Interface Sci 249:100–133

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors express their gratitude to all the members in the laboratory of Molecular Pathology College of Veterinary Medicine, Northeast Agricultural University for providing advanced equipment and conditions.

Funding

This study is funded by grants from National Natural Science Foundation of China (32472996) to Dexing Ma; Heilongjiang Natural Science Foundation (LH2022C035) to Dexing Ma.

Author information

Author notes

  1. Hang Chen and Wenjing Zhi are co-first authors.

Authors and Affiliations

  1. College of Veterinary Medicine, Northeast Agricultural University, Harbin, China

    Hang Chen, Wenjing Zhi, Bingrong Bai, Zhipeng Jia, Rui Kong, Qiuju Liu, Biao Wang & Dexing Ma

  2. Bristol Veterinary School, University of Bristol, Langford, Bristol, UK

    Faisal R. Anjum

  3. College of Food Science, Northeast Agricultural University, Harbin, China

    Chunli Ma

  4. Heilongjiang Provincial Key Laboratory of Pathogenic Mechanism for Animal Disease and Comparative Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, China

    Dexing Ma

Authors
  1. Hang Chen
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Wenjing Zhi
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Bingrong Bai
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Faisal R. Anjum
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Zhipeng Jia
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Rui Kong
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Qiuju Liu
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Biao Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Chunli Ma
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. Dexing Ma
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

HC: conceptualisation, methodology, roles/writing-original draft. WZ: data curation, software, formal analysis. BB: investigation. FRA: revision. ZJ: software. RK: data curation. QL: methodology. BW: formal analysis. CM: conceptualisation, validation, supervision, writing–review and editing. DM: funding acquisition, project administration, resources, writing–review and editing. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Chunli Ma or Dexing Ma.

Ethics declarations

Ethics approval and consent to participate

This study was performed according to the National Guidelines for Experimental Animal Welfare. Animal experiments were performed based on the regulations of the Ethical Committee for animal sciences in Northeast Agricultural University. The protocol was approved by the Animal Ethics Committee of Northeast Agricultural University (NEAUEC20220335).

Competing interests

The authors declare that they have no competing interests.

Additional information

Handling editor: Frank Katzer.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1

. Prediction of secondary structure of EtCab protein. h means alpha-helix; c means random coi; t means beta-turn; e means extended strand.

Additional file 2

. Optimization evaluation of EtCab model. The red areas correspond to the “core” regions representing the most favourable combinations of phi-psi values.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, H., Zhi, W., Bai, B. et al. Impact of sodium alginate hydrogel containing bacteriophage peptides that specifically bind to the EtCab protein on the inhibition of Eimeria tenella infection. Vet Res 56, 18 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13567-024-01425-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13567-024-01425-4

Keywords

Veterinary Research

ISSN: 1297-9716