
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00735-1
10.1016/j.psj.2024.104156
104156
IMMUNOLOGY, HEALTH AND DISEASE
Effect of orally administered B. subtilis-cNK-2 on growth performance, immunity, gut health, and gut microbiome in chickens infected with Eimeria acervulina and its potential as an alternative to antibiotics1
Wickramasuriya Samiru S. *
Park Inkyung *
Lee Youngsub *
Richer Luciana M. †
Przybyszewski Chris †
Gay Cyril G. ‡
van Oosterwijk Jolieke G. †
Lillehoj Hyun S. hyun.lillehoj@usda.gov
*2
⁎ Animal Bioscience and Biotechnology Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA
† US Biologic, Inc., Memphis, TN 38103, USA
‡ Office of National Program-Animal Health, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705, USA
2 Corresponding author: hyun.lillehoj@usda.gov
05 8 2024
11 2024
05 8 2024
103 11 10415619 4 2024
29 7 2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
This study investigated the best oral delivery strategy (gavage or feed) for the B. subtilis expressing the chicken anti-microbial peptide cNK-2 (B. subtilis-cNK-2) in comparison to monensin, in chickens challenged with Eimeria acervulina (E. acervulina). A total of 120 broiler chickens were randomly allocated into 5 treatment groups in a completely randomized design: 1) uninfected chickens fed with basal diet (NC), 2) E. acervulina-infected chickens fed a basal diet (PC), 3) E. acervulina-infected chickens fed a basal diet supplemented with 90 mg monensin/kg feed (MO), 4) E. acervulina-infected chickens fed a basal diet and orally gavaged with B. subtilis-cNK-2 at 1 × 1010 cfu/d (CNK-O), and 5) E. acervulina-infected chickens fed a basal diet mixed with B. subtilis-cNK-2 at 1 × 1010 cfu/kg feed (CNK-F). The challenge consisted of 5,000 sporulated E. acervulina oocysts through oral gavage on d 15. Body weights were measured on d 7, 14, 21, and 23. Duodenal tissue and digesta samples were collected at 6 d postinfection (dpi) to assess the gut integrity, oxidative stress, mucosal immunity, and the gut microbiome. Fecal samples were collected from 6 to 8 dpi to enumerate the oocyst shedding. Chickens in the CNK-O group showed improved (P < 0.05) growth performance, gut integrity, and mucosal immunity compared to PC, comparable to chickens in the MO group. Chickens in the MO, CNK-F, and CNK-O treatment groups all showed lower (P < 0.05) oocyst shedding compared to PC chickens. Moreover, distinct cytokine profile, oxidative stress measures, tight junction proteins, and shifts in the gut microbiome with associated functional changes were observed in all challenge groups. In conclusion, we showed that the oral administration of B. subtilis-cNK-2 improved growth performance, enhanced local protective immunity, and reduced fecal oocyst shedding in broiler chickens infected with E. acervulina, demonstrating potential use of B. subtilis-cNK-2 as an alternative to antibiotics to protect chickens against coccidiosis.

Key words

NK-lysin
Bacillus subtilis
coccidiosis
gut health
alternative to antibiotic
==== Body
pmcINTRODUCTION

Extensive use of in-feed antibiotics in commercial poultry production was considered a turning point for the modern poultry industry until antimicrobial resistance-causing superbugs were discovered in humans and animals (Lillehoj et al., 2018). Antimicrobial resistance has since been declared a significant emerging threat to global public health and food security by the Food and Agriculture Organization (Wall et al., 2016). Consequently, legislative, and public pressure on antibiotic usage led to the imposition of a ban on antibiotics as growth promoters in livestock production. In 2023, to support this limit to the judicious use of antibiotics in animals, The United States Food and Drug Administration (FDA) Center for Veterinary Medicine (CVM) limited the antibiotics to use only under the oversight of licensed veterinarians (Sarkar and Okafor, 2023). While this is a necessary change, supporting the fight against antimicrobial resistance (AMR), imposed in-feed antibiotic restrictions lead to an increase in the occurrence of gut infections such as coccidiosis and necrotic enteritis in chickens. To continue to support the fight against AMR and improve the health status of the animals, there is a timely need to develop novel alternatives to antibiotics to sustain the industry while catering to the growing consumer demand for safe eggs and meat.

During the last few decades, animal scientists have intensively searched for alternatives to antibiotics such as probiotics, prebiotics, synbiotics, essential oils, phytochemicals, antimicrobial peptides, enzymes, and hyperimmune egg antibodies (Lee et al., 2022a). Many of these alternatives demonstrated promising effects on growth and enhanced gut health in broiler chickens. Among these alternatives, antimicrobial peptides (AMPs) emerged as natural evolutionary conserved host defense proteins against a broad range of microorganisms (Kim et al., 2016; Wang et al., 2016). These biological molecules are abundant in hydrophobic cationic residues which facilitate disruption of the bacterial membrane based on their amphipathic nature (Kim et al., 2017). Based on the structural features, AMPs have been characterized into 4 major groups: α-helical, b-hairpin, b-sheet, and extended AMPs in traditional classification (Powers and Hancock, 2003; Huan et al., 2020). Later, new granulysin and NK-lysin classes have been recognized with much larger (74–78 amino acids) and globular 3-dimensional structures (Kim et al., 2017).

Originally, NK-lysin was identified in the natural killer cells of porcine intestinal tissues, and later chicken NK-lysin (cNK-lysin) was discovered in the intestinal lymphocytes of Eimeria-infected chickens (Hong et al., 2006a). The characterization, antimicrobial properties, and immunomodulatory activities of chicken NK-lysin and its derivative cNK-2 peptide were reported previously (Hong et al., 2006a; Hong et al., 2008; Kim et al., 2017). Recently, we reported the clinical success of a novel oral-delivery system using B. subtilis spores expressing a chicken NK-lysin peptide (B. subtilis-cNK-2) as an alternative to antibiotics to mitigate avian coccidiosis in broiler chickens (Wickramasuriya et al., 2021; Wickramasuriya et al., 2023). According to our research, the disturbance of the parasitic membrane and regulation of the inflammatory response constitute the primary underlying mechanisms of action of the chicken NK-lysin peptide. Based on these findings, we conducted the present study to further understand the protective activity of B. subtilis-cNK-2 and to determine the best route for B. subtilis-cNK-2 to reach the intestinal targets with optimum effectiveness to enhance growth performance, gut integrity, and immunity in Eimeria acervulina (E. acervulina)-infected broiler chickens as compared to an industry-standard ionophore. More importantly, we evaluated the local changes in gut microbiome and its functional changes associated with B. subtilis-cNK-2 in this study.

MATERIALS AND METHODS

Experimental Design and Animal Care

Male Ross broiler chickens (Ross 708) were obtained (n = 120) from a local commercial hatchery (Longnecker Hatchery, Elizabethtown, PA) on the day of hatch. Upon arrival, the chickens were randomly assigned to 5 treatment groups after their individual body weights were recorded. Care was taken to ensure that the body weight and weight distribution were similar across all treatment groups during allocation. Each treatment contained 6 replicate cages with 4 chickens each. Chickens were raised in experimental cage units housed in a temperature-controlled closed-house environment. All management practices followed the Ross broiler management handbook (Aviagen, 2018) and complied with the approved animal protocols.

Experimental treatments included: 1) uninfected chickens fed with basal diet (Table 1) (NC), 2) infected chickens fed with basal diet (PC), 3) infected chickens fed with basal diet containing 90 mg monensin/kg feed (MO), 4) infected chickens fed with basal diet and orally administrated (oral gavage) with B. subtilis-cNK-2 at 1 × 1010 cfu/d (CNK-O), and 5) infected chickens fed with a basal diet supplemented with B. subtilis-cNK-2 at 1 × 1010 cfu/kg (CNK-F).Table 1 Ingredient composition of basal diet (as-fed basis).

Table 1Ingredients	Amount, %	
Corn	55.78	
Soybean meal	37.03	
Soybean oil	2.97	
Dicalcium phosphate	1.8	
Calcium carbonate	1.51	
Salt	0.38	
Vitamin mix1	0.22	
Mineral mix	0.15	
DL-Methionine	0.1	
Choline-chloride, 60%	0.06	
Total	100	
Calculated values	
ME, Kcal/kg	3500	
Crude protein, %	24	
Calcium, %	1.2	
Ava. Phosphorus, %	0.51	
Lysine, %	1.4	
Methionine, %	0.49	
Met+Cys, %	0.8	
1 Vitamin mixture provided the following nutrients per kg of diet: vitamin A, 2,000 IU; vitamin D3, 22 IU; vitamin E, 16 mg; vitamin K, 0.1 mg; vitamin B1, 3.4 mg; vitamin B2, 1.8 mg; vitamin B6, 6.4 mg; vitamin B12, 0.013 mg; biotin, 0.17 mg; pantothenic acid, 8.7 mg; folic acid, 0.8 mg; niacin, 23.8 mg.

2Mineral mixture provided the following nutrients per kg of diet: Fe, 400 mg; Zn, 220 mg; Mn, 180 mg; Co, 1.3 mg; Cu, 21 mg; Se, 0.2 mg.

From d 14 up to d 18, B. subtilis-cNK-2 was administered via oral gavage (total volume of 1 mL per chicken) to the chickens in the CNK-O treatment group (Figure 1). On d 15, all chickens except the uninfected control (NC) chickens were infected with freshly propagated E. acervulina (ARS Beltsville strain #12) oocysts (5,000/chicken) through oral gavage. The B. subtilis-cNK-2 peptide used in this experiment was produced by US Biologic, Inc. (Memphis, TN) as described previously (Wickramasuriya et al., 2021).Figure 1 Schematic outline of the experimental design. Experimental feed included: 1) basal diet (for NC, PC, and CNK-O), 2) basal diet containing 90 mg monensin/kg feed (MO), 3) basal diet supplemented B. subtilis-cNK-2 at 1 × 1010 cfu/kg (CNK-F). Chickens in CNK-O treatment were oral gavage with B. subtilis-cNK-2 at 1 × 1010 cfu/d from d 14 to d 18.

Figure 1

Chickens had ad libitum access to feed and fresh clean water until the end of the 24-d experimental period. All experimental procedures were approved by the Beltsville Agricultural Research Center Small Animal Care Committee (Animal Protocol No. 20-002).

Body Weight Measurement

Each chicken was weighed separately on d 1, 7, 14, 21, and 24 to determine body weight and calculate the average daily weight gain.

Sample Collection

On d 21 (6 d postinfection; dpi), 6 chickens from each treatment group were randomly selected for sample collection. After humanely sacrificing chickens by cervical dislocation, duodenum samples were obtained. Digesta samples were collected aseptically into plastic external thread cryogenic vials, immersed in liquid nitrogen, and stored at -80°C for microbiome analysis. After making a longitudinal incision, the duodenal mucosa was carefully scraped using a tissue scraper. The collected mucosal samples were then stored in RNA stabilization solution (RNAlater solution, Invitrogen Corporation, Carlsbad, CA) at -20°C.

Oocyst Shedding

To quantify fecal oocyst shedding, fecal samples were collected from each cage from d 21 (6 dpi) to d 23 (8 dpi). The fecal samples were processed following the reference method by Lee et al. (2018). Specifically, feces from each cage were homogenized with 3 liters of fresh water. Two subsamples from each homogenized mixture were then placed into 50 mL tubes for oocyst counting. Prior to counting, serial dilutions were performed for each sample. Three scientists independently counted the oocysts using a McMaster counting chamber and the sodium chloride flotation technique (Lee et al., 2018). The total oocyst shedding per chicken was calculated using the formula provided by Wickramasuriya et al. (2023).

RNA Extraction and qRT-PCR

All RNA extraction was followed the same protocol as published in our previous publication (Wickramasuriya et al., 2023). Briefly, total RNA was extracted from duodenum mucosa using TRIzol reagent (Invitrogen, Carlsbad, CA) followed by DNase digestion as described previously (Park et al., 2020). The RNA quality and the concentration were determined by NanoDrop spectrophotometer (NanoDrop One; Thermo Scientific, Wilmington, DE). QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) was used to synthesis the cDNA following the manufacturer's instructions. The samples were triplicated and analyzed using Applied Biosystems QuantStudio 3 Real-Time PCR Systems (Life Technologies, Carlsbad, CA). The set PCR conditions were: denaturation (95°C for 2 min) followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.

The gene expression levels of tight junction proteins in duodenum mucosa samples, such as mucin 2 (MUC-2), zonula occludens 1 (ZO-1), and occludin (OCLN) and antioxidant markers, including superoxide dismutase type 1 (SOD-1), Heme oxygenase 1 (HMOX-1), and catalase (CAT) were investigated. The cytokine gene expression levels of duodenum mucosa including Interleukin (IL)-2, IL-22, IL-6, and interferon-gamma (IFN-γ), were also quantified. All oligonucleotide primer sequences used in this experiment are shown in Table 2. Relative gene expression levels were calculated using the logarithmic-scaled threshold cycle (Ct) values in the 2−∆∆Ct method and normalized to those of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reference gene.Table 2 Quantitative real-time PCR oligonucleotide primer sequences.

Table 2Target gene	Primer sequence	Accession No.	
GAPDH	F 5′-GGTGGTGCTAAGCGTGTTAT-3′	K01458	
R 5′-ACCTCTGTCATCTCTCCACA-3′	
OCLN	F: 5′-GAGCCCAGACTACCAAAGCAA-3′	NM205,128.1	
R: 5′-GCTTGATGTGGAAGAGCTTGTTG-3′	
ZO-1	F: 5′-CCGCAGTCGTTCACGATCT-3′	XM01,527,8981.1	
R: 5′-GGAGAATGTCTGGAATGGTCTGA-3′	
MUC-2	F: 5′-GCCTGCCCAGGAAATCAAG-3′	NM0,013,18434.1	
R: 5′-CGACAAGTTTGCTGGCACAT-3′	
HMOX-1	F 5′-CTGGAGAAGGGTTGGCTTTCT-3′	NM205344	
R 5′-GAAGCTCTGCCTTTGGCTGTA-3′	
SOD-1	F 5′-ATTACCGGCTTGTCTGATGG-3′	NM205064.1	
R 5′-CCTCCCTTTGCAGTCACATT-3′	
CAT	F 5′-ACTGCAAGGCGAAAGTGTTT-3′	NM001031215.1	
R 5′-GGCTATGGATGAAGGATGGA-3′	
IL-2	F: 5′-TACAGATAACTGGGACACTG-3′	NM_204153.1	
R: 5′-GTCTCAGTTGGTGTGTAGAG-3′	
IL-6	F: 5′-CAAGGTGACGGAGGAGGAC-3′	NM_204628	
R: 5′-TGGCGAGGAGGGATTTCT-3′	
IL-22	F 5′-ACCCGTATGCTGAGGATGTGG-3′	NM_001199614.1	
R 5′-CTTGTTCCCTCCCTTCTTTGG-3′	
IFN-γ	F: 5′-AGCTGACGGTGGACCTATTATT-3′	NM_205149.1	
R: 5′-GGCTTTGCGCTGGATTC-3′	
Abbreviations: GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; OCLN, Occludin; ZO-1, Zonula occludens-1; MUC-2, Mucin 2; HMOX-1, Heme oxygenase 1; SOD-1, Superoxide Dismutase 1; CAT, Catalase; IL, Interleukin; IFN-γ, Interferon-gamma; F, forward primer; R, reverse primer

Microbial 16S rRNA Analysis of Digesta

Samples of collected digesta were sent to outside laboratory (EzBiome, Gaithersburg, MD) for DNA extraction, preparation of the 16S rRNA gene library, and high-throughput sequencing. The DNA extraction was carried out, and the concentration of genomic DNA was measured using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA). The 16S rRNA V3-V4 regions within the ribosomal transcript were amplified using the primer pair (Illumina-F: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and Illumina-R: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) which contains the gene‐specific sequences and Illumina adapter overhang nucleotide sequences. Amplicon polymerase chain reaction (PCR) was performed to amplify the template out of input DNA samples as described elsewhere (Brumfield et al., 2022). In brief, a 25 μL PCR reaction contained 12.5 ng of input DNA, 12.5 μL of 2× KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, MA), and 5 μL of each primer at a 1 μM concentration. The PCR process began with an initial denaturation at 95°C for 3 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, ending with a final extension at 72°C for 5 min. The PCR products were purified using Mag-Bind RxnPure Plus magnetic beads (Omega Bio-tek, Norcross, GA). A subsequent index PCR, which followed the same conditions, added barcodes and sequencing adapters with 8 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, finishing with a final elongation at 72°C for 5 min. The libraries were then normalized, pooled, assessed using an Agilent 2200 TapeStation, and sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) using a 500-cycle (2 × 250 bp paired-end) run.

16S Amplicon Taxonomic Assignment

Taxonomic profiling of the 16S sequencing data was performed by uploading the paired-end reads to the EzBioCloud microbiome taxonomy profiling platform (www.ezbiocloud.net), following the method outlined by Yoon et al. (2017). In brief, the EzBioCloud cloud application filters out low-quality sequences based on read length (<80 bp or >2,000 bp) and average Q values below 25. Denoising and extraction of non-redundant reads were performed using the DUDE-Seq software. The UCHIME algorithm (Edgar et al., 2011) was applied against the EzBioCloud 16S chimera-free database to remove chimeric sequences and correct inflated diversity from the 16S rRNA sequencing methodology. Taxonomic assignment was conducted using the USEARCH program to identify and calculate sequence similarities of the query single-end reads against the EzBioCloud 16S database. Sequencing reads were clustered into operational taxonomic units (OTU) at 97% sequence similarity using the UPARSE clustering algorithm (Edgar, 2013). Each sample's reads were clustered into multiple OTUs using the UCLUST tool with the aforementioned cutoff values. For the EzBioCloud 16S-based Microbiome Taxonomic Profiling (MTP) pipeline, the PICRUSt algorithm was used to estimate the functional profiles of the microbiome identified through 16S rRNA sequencing.

Calculations and Statistical Analysis

Obtained data was analyzed using a generalized linear model (GLM) procedure with one-way ANOVA in SPSS software (Version 24; IBM SPSS 2016, Armonk, NY). The individual chicken was considered the experimental unit for statistical analysis. The results are given as least-squares means and pooled SEM. P-values < 0.05 were considered as significant. When the P-value between treatments was less than 0.05, homogeneous subsets were evaluated by Duncan's multiple range test in SPSS.

Microbiome data calculation and analysis were performed on the EzBiomeCloud platform (Yoon et al., 2017). Briefly, microbial richness was investigated using abundance-based coverage estimates (ACE), species richness (for microbial diversity) using Chao1, and taxonomy using the number of OTUs found in the MTP index. The Shannon, Simpson, and Phylogenetic indices were applied to estimate the α-diversity for each group using the Wilcoxon rank-sum test. Beta diversity was calculated with Jansen-Shannon distances based on the taxonomic abundance profiles. Permutational multivariate analysis of variance (PERMANOVA) was applied to measure the statistical significance of β-diversity. Different groups were clustered with principal component analysis (PCA) based on the Jaccard distance metric. Linear discriminant analysis Effect Size (LefSe) analysis was carried out using the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) pipeline. PICRUSt output at pathway levels of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was then used to predict the functional biomarker discovery at a threshold of LDA score >2 with significant p < 0.05.

RESULTS

Body Weight Measurement

No body weight difference (d 1–14: P > 0.05) was observed among treatment groups before E. acervulina infection (Table 3). After infection, at d 21 (6 dpi), E. acervulina-infected chickens in PC, CNK-O, and CNK-F showed lower (P < 0.05) body weights compared to the NC and MO treatments. However, chickens in both MO and CNK-O showed similar (P > 0.05) body weights at d 24 (9 dpi), while chickens in PC and CNK-F treatment groups showed lower (P < 0.05) body weights compared to CNK-O chickens.Table 3 Growth performance of Eimeria acervulina-infected chickens fed with antibiotics and chicken NK-lysin1.

Table 3Item	NC	PC	MO	CNK-O	CNK-F	SEM	P-value	
Body weight, g	
D 7	197.97	212.30	213.88	203.25	208.98	2.191	0.120	
D 14	510.07	519.36	516.53	514.88	515.67	3.558	0.950	
D 21 (6dpi)	1009.62a	884.80b	1000.33a	934.08b	900.32b	12.114	0.001	
D 24 (9dpi)	1233.45a	1121.18c	1209.43ab	1136.07b	1104.48c	14.183	0.004	
Average daily gain, g/bird/d	
D 1-7	20.88	22.86	23.08	21.65	22.45	0.322	0.169	
D 7-14	44.47	43.10	43.40	44.08	44.27	0.371	0.748	
D 1-14	32.73	33.34	33.17	33.12	33.38	0.267	0.940	
D 14-21(-1 - 6 dpi)	79.85a	60.02c	80.62a	72.62ab	64.60bc	2.078	0.002	
D 21-24 (6 - 9dpi)	89.23a	71.98b	71.98b	66.97b	67.14b	2.119	0.001	
D 14-24 (-1 - 9dpi)	82.48a	67.08c	77.52ab	71.43b	66.03c	1.641	0.003	
1 All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 4 dpi. NC = Uninfected control birds fed with basal diet; PC = Infected control birds fed with basal diet; MO = Infected chickens fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chickens fed with basal diet and oral administrated Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chickens fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed.

a∼c Means in the same row with different superscripts differ (P < 0.05) and the difference was evaluated by PDIFF option in SAS when P-value between treatments was less than 0.05.

Similar to the body weight data, no ADG difference (P > 0.05) was observed among treatment groups from d 1 to d 14. However, the ADG of chickens in the MO and CNK-O groups were higher (P < 0.05) than those of PC chickens from d 14 to 21 (-1 to 6 dpi). Likewise, chickens in the MO and CNK-O showed higher ADG (p < 0.05) compared to other infected chickens from d 14 to 24 (-1 to 9 dpi).

Fecal Oocyst Shedding

Fecal oocyst shedding of E. acervulina-infected chickens treated with B. subtilis-cNK-2 is presented in Figure 2. As expected, chickens from all groups infected with E. acervulina showed higher (P < 0.05) fecal oocyst shedding as opposed to no shedding in the chickens in NC. Chickens from all treatment groups showed significantly lower oocyst shedding compared to PC. Chickens in the MO group showed the lowest oocyst shedding followed by CNK-F and CNK-O groups with all differences were statistically significant (P < 0.05).Figure 2 Fecal oocyst shedding of Eimeria acervulina-infected chickens fed with antibiotics and chicken NK-lysin. All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 3 dpi. NC = Uninfected control birds fed with basal diet; PC = Infected control birds fed with basal diet; MO = Infected chickens fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chickens fed with basal diet and oral administrated Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chickens fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed. Each bar represents the mean ± SEM (n = 6). Fecal samples were collected from 6 to 8 d postinfection to enumerate the oocyst shedding.a∼e Bars with different superscripts differ (P < 0.05) significantly.

Figure 2

Mucosal Cytokine Gene Expression

At 6 dpi, cytokine gene expression in the duodenal mucosa of chickens was analyzed to evaluate the host immune responses between treatment groups (Figure 3). The chickens in the PC treatment group showed elevated (P < 0.05) IL-2, IL-6, IL-22, and IFN-γ expression levels compared to the chickens in the uninfected NC group. Compared to the PC treatment group, broiler chickens in MO and CNK-O expressed lower (P < 0.05) cytokine levels in duodenal mucosa. The expression levels of IL-2, IL-6, IL-22, and IFN-γ in the mucosa were elevated (P < 0.05) in CNK-F chickens compared to their CNK-O counterparts.Figure 3 Cytokine gene expression in duodenal mucosa of Eimeria acervulina-infected chickens fed with antibiotics and chicken NK-lysin. All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 3 dpi. NC = Uninfected control birds fed with basal diet; PC = Infected control birds fed with basal diet; MO = Infected chickens fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chickens fed with basal diet and oral administrated Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chickens fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed. Each bar represents the mean ± SEM (n = 6). a∼d Bars with different superscripts differ (P < 0.05) significantly.

Figure 3

Antioxidant Gene Expression

As shown in Figure 4, all chickens infected with E. acervulina (PC, MO, CNK-O, CNK-F) showed down-regulated (P < 0.05) expression levels of SOD-1, CAT, and HMOX-1 genes in the duodenum compared to chickens in the NC group. Among the chickens in the infected groups, no significant difference in antioxidant gene expression (P > 0.05) was observed on 6 dpi.Figure 4 Antioxidant gene expression in duodenal mucosa of Eimeria acervulina-infected chickens fed with antibiotics and chicken NK-lysin. All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 3 dpi. NC = Uninfected control birds fed with basal diet; PC = Infected control birds fed with basal diet; MO = Infected chickens fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chickens fed with basal diet and oral administrated Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chickens fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed. Each bar represents the mean ± SEM (n = 6). a∼d Bars with different superscripts differ (P < 0.05) significantly.

Figure 4

Expression of Tight Junction and Mucin Genes

Tight junction and mucin gene expression of E. acervulina-infected chickens fed B. subtilis-cNK-2 is presented in Figure 5. All chickens in the infected treatment groups (PC, MO, CNK-O, CNK-F) showed down-regulated (P < 0.05) expression levels of ZO-1, OCLN, and MUC-2 genes in the duodenum compared to chickens in the NC group. No significant difference in tight junction and mucin gene expression (P > 0.05) was observed among infected treatment groups.Figure 5 Tight junction and mucin gene expression in duodenal mucosa of Eimeria acervulina-infected chickens fed with antibiotics and chicken NK-lysin. All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 3 dpi. NC = Uninfected control birds fed with basal diet; PC = Infected control birds fed with basal diet; MO = Infected chickens fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chickens fed with basal diet and oral administrated Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chickens fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed. Each bar represents the mean ± SEM (n = 6). a∼d Bars with different superscripts differ (P < 0.05) significantly.

Figure 5

Taxonomic Composition

The phylum, order, family, and genus level composition as well as the most abundant bacterial community, in E. acervulina-infected chickens treated with B. subtilis-cNK-2 are presented in Figures 6 and 7, respectively. At the phylum level, Firmicutes are the most abundant phyla for chickens in all treatment groups making up more than 90% of the bacterial community. Looking into NC and PC treatments, a similar (99.18 vs. 99.75%) relative abundance of Firmicutes was observed. In MO chickens, lower Firmicutes abundance (90.67%) and higher Proteobacteria (5.75%) and Bacteroidetes (2.89%) were observed compared to NC, PC, and CNK-O chickens. Similar to MO, CNK-F chickens showed an elevated Proteobacteria (5.75%) abundance. At the level of order, PC chickens showed higher Lactobacillales abundance (92.75%) compared to NC chickens (47.45%). With the increased Lactobacillales, Clostridiales tend to lower in PC (6.86%) compared to the NC (50.42%) group. However, Clostridiales tend to increase (61.67, 24.16, and 12.71% respectively) in MO, CNK-O, and CNK-F chickens restoring the Lactobacillales abundance. Moreover, Enterobacterales was detected in MO (5.6%) and CNK-F (3.01%) groups. A similar pattern was observed at the family level. To further understand the bacterial composition, the genus level was explored. The most abundant microbial genera in NC chickens were Lactobacillus (43.62%), Arthromitus (39.11%), and Ruminococcus_g4 (4.82%). With the E. acervulina infection, the abundance of Lactobacillus increased (43.62%), while Arthromitus was reduced (5.54%) and Ruminococcus_g4 disappeared in the PC group. However, MO restored the Arthromitus (47.63%) and Ruminococcus_g4 (4.2%) while reducing the Lactobacillus (23.71%) abundance. Furthermore, Escherichia (5.39%) and Bacteroides (2.89%) were detected in MO chickens compared to NC and PC groups. In comparison to the PC group, CNK-O, and CNK-F chickens tend to show lower Lactobacillus (75.25% and 80.99% respectively) while increasing Arthromitus abundance (20.42% and 10.26% respectively). Asaccharospora (2.8% and 1.49% respectively) were detected only in the CNK-O and CNK-F groups.Figure 6 Taxonomic composition of Eimeria acervulina-infected chickens fed with antibiotics and chicken NK-lysin. All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 3 dpi. NC = Uninfected control birds fed with basal diet; PC = Infected control birds fed with basal diet; MO = Infected chickens fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chickens fed with basal diet and oral administrated Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chickens fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed.

Figure 6

Figure 7 The most abundant bacterial community composition of Eimeria acervulina-infected chickens fed with antibiotics and chicken NK-lysin. (A) The heatmap depicts the changes of each phylum and genus with respect to treatment group. (B) Six most abundant bacterial genera detected across MTP set. All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 3 dpi. NC = Uninfected control birds fed with basal diet; PC = Infected control birds fed with basal diet; MO = Infected chickens fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chickens fed with basal diet and oral administrated Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chickens fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed.

Figure 7

Microbial Diversity

The most abundant bacterial communities and the bacterial diversity were evaluated between treatment groups using metrics for abundance (Figure 7) and diversity (Figure 8). At the phylum level, Firmicutes dominate across all treatment groups, while Proteobacteria are notably abundant in the MO and CNK-F chickens (Figure 7A). Following Eimeria infection, there is an increase in Lactobacillus abundance, coupled with a decrease in Arthromitus abundance in all infected chickens except those in the MO group (Figure 7B). According to the Wilcoxon rank-sum test, a higher (P < 0.05) Shannon index was observed in MO chickens compared to the PC group. Moreover, phylogenetic diversity was higher (P < 0.05) in MO chickens compared to other treatments. However, no difference (P > 0.05) in Simpson diversity was observed between treatment groups. Similar to diversity, species richness indexes including OTUs, Ace, and Chao1 were higher (P < 0.05) for MO chickens compared to other treatments (Figure 9). Furthermore, higher (P < 0.05) species richness was observed in CNK-O and CNK-F treatments compared to PC, whereas only the CNK-F treatment induced higher (P < 0.05) richness compared to NC in all 3 indexes.Figure 8 Alpha diversity index analysis of digesta of Eimeria acervulina-infected broiler chickens (6 d postinfection) by Shannon, Simpson and phylogenetic diversity index. All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 3 dpi. NC = Uninfected control chicken fed with basal diet; PC = Infected control chicken fed with basal diet; MO = Infected chicken fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chicken fed with basal diet and oral administrated (d 14 to d 18) Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chicken fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed. Significant results (P < 0.05) are marked as (――).

Figure 8

Figure 9 Species richness of digesta of Eimeria acervulina-infected broiler chickens (6 d postinfection) by OTUs, Ace and Chao1 index. All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 3 dpi. NC = Uninfected control chicken fed with basal diet; PC = Infected control chicken fed with basal diet; MO = Infected chicken fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chicken fed with basal diet and oral administrated (d 14 to d 18) Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chicken fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed. Significant results (P < 0.05) are marked as (――).

Figure 9

To capture the variability of distinguishable colonizing bacterial community profiles of the treatment groups, the Bray–Curtis dissimilarity index was calculated and used for Principal Component Analysis (Figure 10). A drastic microbiome shift (P < 0.05) was observed in E. acervulina-challenged chickens either untreated (PC) or treated with monensin (MO) compared to the unchallenged chickens (NC). Chickens treated with cNK-2, either through oral gavage (CNK-O) or in the feed (CNK-F), showed clustering overlapping with both the NC and PC groups. Overall, the treatment groups formed clusters that depicted the distinct microbial shifts in the microbiome as a result of the E. acervulina infection, with the B. subtilis-cNK-2 groups clustering separately from the monensin-treated group.Figure 10 Principal coodinates analysis of percentage read counts for bacteria in the microbiome grouped by species present in Eimeria acervulina-infected broiler chickens (6 d postinfection). All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 3 dpi. NC = Uninfected control chickens fed with basal diet; PC = Infected control chickens fed with basal diet; MO = Infected chickens fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chickens fed with basal diet and oral administrated (d 14 to d 18) Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chickens fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed.

Figure 10

Functional Biomarker Discovery

LefSe analysis revealed that 18 KEGG pathways were significantly affected by the experimental treatments in this study (Figure 11). Among functional pathways, most of the functions were enriched when the chickens were infected with E. acervulina. Whereas most of those pathways showed a lower enrichment in monensin-treated chickens, enrichment in B. subtilis-cNK-2 was more comparable to the untreated challenged (PC) chickens. Among all identified functions, “Flagellar assembly” (ko01110) was the only one that did not enrich with the E. acervulina infection.Figure 11 Predicted functions of KEGG pathways affected by Eimeria acervulina-infected broiler chickens (6 d postinfection). All chickens except NC were infected by oral gavage at d 15 with 5,000 oocysts/chicken of E. acervulina. Bacillus subtilis was administrated (1010 cfu/d) to CNK-O treatment group by oral gavage at - 1 to 3 dpi. NC = Uninfected control chicken fed with basal diet; PC = Infected control chicken fed with basal diet; MO = Infected chicken fed with diet containing 90 mg monensin/kg feed; CNK-O = Infected chicken fed with basal diet and oral administrated (d 14 to d 18) Bacillus subtilis expressing cNK-2 at 1010 cfu/d; CNK-F = Infected chicken fed with diet containing Bacillus subtilis expressing cNK-2 at 1010 cfu/kg feed.

Figure 11

DISCUSSION

We have previously reported the protective effects of orally delivered stable B. subtilis spores containing a modified cNK-lysin peptide against E. acervulina infection in broiler chickens (Wickramasuriya et al., 2021). Expanding upon these findings, our latest research showed advantageous impacts of orally administered B. subtilis-cNK-2 on the gut microbiome, as well as reduced oxidative stress, and increased immunity in E. acervulina-infected broiler chickens (Wickramasuriya et al., 2023). In this current study, we conducted a comparative analysis between oral- and feed-based delivery methods for B. subtilis-cNK-2 and an industry-standard ionophore, to assess their respective influences on the growth performance, oocyst shedding, and gut health of E. acervulina-challenged broiler chickens.

In the current study, we confirmed that E. acervulina infection lowered the body weight and reduced weight gain of the broiler chickens. Similarly, impaired growth performance and higher mortality were seen in commercial chickens (Lee et al., 2022a; Wickramasuriya et al., 2022). Even under the experimental conditions, significant growth retardation was reported in chickens, as a result of Eimeria replication within the intestinal epithelial lining causing lesions and nutrient malabsorption (Leung et al., 2019; Wickramasuriya et al., 2021; Park et al., 2022). The data presented here provide further evidence that the lowered growth performance in E. acervulina-infected chickens likely results from higher oocyst proliferations, lower tight junction permeability, lower antioxidant capacities, elevated cytokine expressions, and unfavorable gut microbiome changes, and that can be mitigated using oral delivery of cNK-2 peptide.

We observed that E. acervulina-infected chickens treated with monensin showed a higher growth performance compared to untreated, challenged chickens, confirming findings from other groups (Talghari et al., 2020; Park et al., 2022) that monensin attenuates many of the negative effects of coccidiosis. Similar to monensin treatment, higher body weight, and weight gain are achieved using oral cNK-2 peptide, indicating its potential as an effective alternative to antibiotics and confirming the findings from our previous studies (Wickramasuriya et al., 2021; Wickramasuriya et al., 2023). The effect on growth performance was higher in the CNK-O treated chickens compared to the CNK-F treated chickens. Therefore, these results suggest that the lower growth performance of CNK-F vs CNK-O might be due to the triggered cytokine gene expression of the chickens in that group. In support of this notion, Zhou et al. (2020) explained the negative correlation between growth performance and heightened cytokine expression in broiler chickens. By regulating hypothalamic pituitary outflow, cytokines such as IL-1β and IL-6 may modulate protein, carbohydrate, and fat metabolism while regulating the feed intake and feeding behaviors of the chickens (Zhou et al., 2020). Moreover, it may be necessary to reassess the dosage and duration of B. subtilis-cNK-2 treatment in the CNK-F feed to mitigate disease response and improve protection given the chickens' feed intake.

E. acervulina challenge in this study led to fecal oocyst shedding, indicating the invasion, establishment, and propagation of Eimeria in the chicken intestine, as has been reported previously (Lee et al., 2013; Lillehoj et al., 2014; Wickramasuriya et al., 2021). An observed lower oocyst shedding in the MO group depicts the long-known positive effect of monensin which has been used to control avian coccidiosis (Bozkurt et al., 2016). Different modes of action for monensin to control coccidiosis have been proposed, including vacuolation (by facilitating the transport of monovalent ions and thereby disrupting ion gradients across cell membranes), swelling and eventual bursting of the sporozoites, and interruption of host cell invasion by sporozoites (Chapman et al., 2010; Robinson et al., 2019). Additionally, lower oocyst shedding in chickens treated with monensin has been reported (Bozkurt et al., 2016; Park et al., 2022). Here, in addition to chickens treated with monensin, B. subtilis-cNK-2 administration (when administered either through oral gavage or through the feed) showed significantly lowered fecal oocyst shedding in E. acervulina-infected chickens. The results presented here confirm our previously reported successful delivery of B. subtilis-cNK-2 into the intestinal target sites and its subsequential protective activity against coccidiosis (Wickramasuriya et al., 2021; Wickramasuriya et al., 2023). Interestingly, among B. subtilis-cNK-2 groups, CNK-F chickens showed significantly lower oocyst shedding compared to CNK-O chickens. Presumably, it might be an accumulated effect and/or confounding effect of B. subtilis and cNK-2 peptide at the intestinal sites where coccidia invaded.

An in-depth understanding of the avian immune responses is of utmost importance for developing effective control strategies against coccidiosis. To assess the relevant gene activations, biological effects, receptor interactions, and signal transduction, cytokine assays are being utilized for chicken studies although the avian cytokine repertoire is limited (Lu et al., 2022). Coccidiosis triggers variety of cytokines and chemokines involved in mediating innate and acquired immune responses in chickens (Lillehoj, 1998). Early findings with coccidiosis revealed the significance and importance of pro-inflammatory cytokine IFN-γ, and other T cells producing interleukins, which have an immunomodulatory effect in chickens (Kim et al., 2019). To investigate mucosal immunity, we looked at cytokine gene expression in the duodenum where the most intense epithelial damage occurs following E. acervulina infection. On 6 dpi, we observed the surge of cytokine (IL-2, IL-6, IL-22, and IFN-γ) expression in Eimeria-infected chickens compared to the uninfected chickens illustrating the role of multiple cytokines in initiating anti-parasitic immune responses. This was the same pattern we observed in our previous study with E. acervulina infections (Wickramasuriya et al., 2023) and has also been described in other studies that reported the upregulated cytokine gene expressions with E. maxima (Park et al., 2020, 2021) and E. acervulina (Rochell et al., 2017). This cascade of cytokine expression in Eimeria-infected chickens may also be explained by the gut microbiome changes which will be discussed later on.

Earlier studies from our laboratory showed that pro-inflammatory cytokines are linked with reduced Eimeria replication and anti-inflammatory cytokines play an immunomodulatory role in chickens (Hong et al., 2006b). As observed in the MO chickens, lower mucosal cytokine gene expressions were observed in the CNK-O group, providing a potential explanation for the observed higher growth performance in CNK-O chickens and associated lower oocyst shedding in this group. However, this pattern was not observed in the CNK-F group, where the growth performance was less apparent than in the CNK-O group.

It is well documented that broiler chickens are continuously subjected to oxidative stress conditions as a result of intestinal diseases that disturb the enzymatic antioxidant system in the body (Lee et al., 2020; Wickramasuriya et al., 2022). Imbalances of degradation and production of reactive nitrogen species and reactive oxygen species such as hydrogen peroxide, superoxide anions, and lipid peroxide cause oxidative stress. As reported, oxidative stress is prevented by the inactivation of precursors of free radicals or catalysts with the antioxidant enzymes including heme oxygenase, aflatoxin B1 aldehyde reductase (AFAR), SOD-1, CAT, and glutathione peroxidase (Delles et al., 2014; Lee et al., 2018; Surai et al., 2019). Previous studies demonstrated the impact of coccidiosis on oxidative stress leading to lipid peroxidation, antioxidant insults, and destruction of tight junction barrier function in chickens (Khatlab et al., 2019; Mishra and Jha, 2019). According to Khatlab et al. (2019), Eimeria infection downregulated the expression levels of SOD-1 and CAT activity as a result of cellular damage in the jejunum. Moreover, lower HMOX-1 gene expression in duodenal mucosa and lower CAT and HMOX-1 expression in the spleen were reported in E. acervulina-infected broiler chickens compared to uninfected counterparts (Wickramasuriya et al., 2021). Similarly, our results indicated that E. acervulina-infected chickens significantly downregulated SOD-1, CAT, and HMOX-1 expression levels in the duodenal mucosa compared to uninfected chickens. In this study, no significant difference was observed among E. acervulina-infected treatment groups for SOD-1, CAT, and HMOX-1 expressions. Although it is not significant, it is interesting to see the elevated CAT and HMOX-1 expression in the MO group compared to other infected chickens. Similarly, we also did not observe any B. subtilis-cNK-2 effect on SOD-1 and CAT gene expression in the duodenum mucosa in our previous studies (Wickramasuriya et al., 2021; Wickramasuriya et al., 2023). However, HMOX-1 expression in duodenum mucosa was elevated in the B. subtilis-cNK-2 treated E. acervulina-infected chickens in our previous studies (Wickramasuriya et al., 2021). Although it is ambiguous, this discrepancy might be explained by the bird age and different virulency of the E. acervulina used in different studies.

For a better understanding of the treatment effects on the gut health of the chickens, we investigated the tight junction and mucin gene expressions in the duodenum. The tight junction is an intercellular complex that binds epithelial cells tightly at the apical end to form an intestinal epithelium which plays a vital role in maintaining gastrointestinal health by preventing pathogen translocation (Wickramasuriya et al., 2022). The tight junction complex comprises tight junction proteins that include junctional adhesion protein, occludin, zonula occludens, and claudin (Ulluwishewa et al., 2011). In addition to the intestinal epithelium, the mucosa layer is regulated by the mucin-2 gene expression and provides a first-line protection against the intestinal pathogens by preventing pathogens’ movement to epithelial cells (Sharma et al., 2022). By maintaining intestinal permeability, it enhances gut health and nutrient absorption leading to better growth performance in chickens. In the present study, ZO-1, which is considered an adaptor protein that interacts with junctional components (Tornavaca et al., 2015), and OCLN, which plays a role in tight junction assembly and maintenance (Furuse et al., 1994; Park et al., 2022), were downregulated 6 d after E. acervulina infection showing the damage brought by Eimeria to the epithelial. This finding is consistent with previous research that has demonstrated the downregulation of tight junction gene expression in Eimeria-infected broiler chickens (Santos et al., 2020; Lee et al., 2022b). To prevent further infection and replenishment of the mucosal layer, MUC-2 gene expression was reported to be downregulated during the coccidiosis (Sharma et al., 2022). We hypothesized that chickens in MO and B. subtilis-cNK-2 groups may show enhanced tight junction gene expression showing gut health improvement in support of growth performance. However, patterns of tight junction gene expression were not significantly different from the infected control chickens in this study. The higher virulency of Eimeria used in the present study compared to previous studies may explain the absence of significant difference in the tight junction gene expression in this study.

Understanding the chicken gut microbiome and its changes resulting from experimental treatments is important and aids in describing the effects of these treatments on performance. In chickens, Firmicutes, Proteobacteria, and Bacteroidetes are the most abundant taxa at the phylum level ( Memon et al., 2022; Wickramasuriya et al., 2023). As we previously reported, higher Firmicutes abundance (>90%) was observed in all treatment groups. It is known that Firmicutes and Bacteroidetes are important in gut homeostasis and nutrition absorption in chickens while Proteobacteria plays a key role in the gut health of the host animal by preparing the gut environment for anaerobic bacteria colonization and reducing the redox potential (Shin et al., 2015; Memon et al., 2022). In this study, although we did not observe Bacteroidetes and Proteobacteria abundance in PC and NC groups, Bacteroidetes and Proteobacteria were detected in the MO group, and Proteobacteria were detected in the CNK-F groups reducing the Firmicutes abundance in these groups.

The impact of coccidiosis on gut microbiome changes in broiler chickens has been reported and reviewed previously (Madlala et al., 2021; Memon et al., 2022; Wickramasuriya et al., 2023). With the Eimeria challenge, it was observed that the proliferation of Lactobacillus spp. contributes to reduced gut lesion scores, enhanced mucosal integrity, and heightened immune-modulatory responses (Vieira et al., 2020). It was especially documented that Lactobacilli stimulates pro-inflammatory cytokines surges such as IFN-γ, IL-22, and TNF-α (Vieira et al., 2020). This study also found higher Lactobacillus abundance in PC chickens followed by CNK-F and CNK-O compared to the NC group, and cytokine expression was highest in the PC and CNK-F groups, further supporting the role of Lactobacillus inducing a pro-inflammatory cytokine surge. Moreover, in this study, lower Lactobacillus abundance and lower cytokine expression were observed in the MO treatment group. Similar to our findings, Lu et al., (2008) also reported that the antibiotic supplementation (Bacitracin/Virginiamycin or monensin) reduced Lactobacilli while increasing Clostridia abundance in the ileal segment of broiler chickens. Although the effect of monensin on the gut microbiota in chickens is not well defined, it has been reported that gram-positive bacteria are considered to be susceptible to monensin, and therefore a decrease in the Lactobacillus population is observed in response to treatment, which is in agreement with our results (Danzeisen et al., 2011; Dec et al., 2020; Bilal et al., 2021). According to Díaz-Sánchez et al. (2019), Lactobacillales were associated with the production of bile salt hydrolase and poor weight gain in broilers. In this light, observed lower growth performance in E. acervulina-infected chickens and restoration with monensin is explainable. Another noticeable microbial change in the present study was Clostridiaceae abundance in Eimeria-challenged chickens. There are different species that belong to many different genera which are categorized under this Clostridiaceae family including Clostridium perfringens and Clostridium butyricum. In this study, coccidiosis significantly reduces the relative abundance of Arthromitus genera (segmented filamentous bacteria) which also belongs to the Clostridiaceae family. Similarly, a decreased abundance of Candidatus Arthromitus was observed in the caecal digesta of Eimeria-infected chickens previously (Stanley et al., 2014; Antonissen et al., 2016). The association and importance of these segmented filamentous bacteria on maturations of gut immunity were reported elsewhere (Antonissen et al., 2016). Higher growth performance observed in NC and MO chickens in the present study may also be attributed to higher Lachnospiraceae, Ruminococcaceae abundance via better feed efficiency associated with short-chain fatty acid and plant material degradation as reported to be positively correlated with Lachnospiraceae, Ruminococcaceae, and Erysipelotrichaceae in chickens (Vieira et al., 2020). Similarly, Danzeisen et al., (2011) also reported the monensin effect on Lachnospiraceae and Ruminococcaceae family and its positive correspondence to growth performance in chickens.

According to the present alpha diversity data, E. acervulina infection caused no significant effects on bacterial diversity and species richness indexes. Similarly, we did not observe any alpha diversity changes in the duodenum digesta of E. acervulina-infected chickens in our previous study (Wickramasuriya et al., 2023). In contrast, Memon et al. (2022) observed a decreased Ace, Shannon, and Chao 1 index together with a higher Simpson index in E. tenella-infected chickens. For different Eimeria species, the location of the intestine where coccidia parasites invade and develop may explain these variabilities in gut microbiome populations between experiments. Interestingly, higher diversity and species richness were observed in the MO chickens showing the monensin effect on the chicken gut microbiome, which could be due to the aforementioned effect of monensin on gram-positive bacteria in general and Lactobacilli in particular, with increased microbiome diversity. Nevertheless, some other studies did not observe significant changes in the diversity indexes of monensin-treated chickens (Herrero-Encinas et al., 2020; Vieira et al., 2020). Similar to our findings, Crisol-Martínez et al. (2017) showed an increased in microbiome diversity in zinc bacitracin-treated chickens. In contrast, decreased diversity, species richness, and evenness of the cecal microbiome were reported with antibiotic-treated chickens (Kairmi et al., 2022).

These conflicting observations can be attributed to several experimental factors such as intestinal segment, bird age and maturity, diet, stress conditions, and timing of sampling. The beta diversity based on Bray-Curtis distance revealed that the total microbiome composition was shifted following E. acervulina infection in chickens. In support of these findings, significant microbiome shifts were observed with the Eimeria-infected chickens in previous studies (Bortoluzzi et al., 2019; Vieira et al., 2020; Wickramasuriya et al., 2023). In chickens treated with monensin, closer clustering and significant deviation of the microbiome in MO chickens compared to PC was observed. Suggesting a holistic protective effect and healthier gut colonization mediated by cNK-2, significant reassembling of gut microbiome towards NC was observed in CNK-O and CNK-F chickens. Similar finding was seen in our previous study with E. acervulina-infected chickens (Wickramasuriya et al., 2023).

To better understand the functional changes in the coccidiosis-induced microbial population, predictive functional analysis was performed. As observed in this study, many of the predicted functions were enriched with the E. acervulina challenge. In the literature, carbohydrate, amino acid, Glycolysis/Gluconeogenesis, and sucrose metabolism pathways are reported to be the most affected when chickens are infected with Eimeria spp (Bortoluzzi et al., 2019, Cai et al., (2022), and this study showed that in untreated chickens and chickens treated with oral NK lysin, these pathways were indeed upregulated. Interestingly, E. acervulina infection significantly increased the enrichment of the quorum sensing pathway in chickens, however, MO chickens showed less enrichment in the quorum sensing pathway supporting previous reports that the immune response against parasites relies on the quorum sensing pathway (Cai et al., 2022), which may be suppressed by monensin treatment.

CONCLUSIONS

This study shows the effectiveness of B. subtilis-cNK-2 as a novel alternative to antibiotics in the mitigation of the negative effects of coccidiosis in broiler chickens. Oral administration of B. subtilis-cNK-2 resulted in enhanced growth performance, lower oocyst production, immune modulation, and alteration of the gut microbiome, providing a good alternative to antibiotics, as shown here in comparison to Monensin. Moreover, it re-confirmed our previous results and supports the potential beneficial effects of orally delivered B. subtilis-cNK-2 as an effective alternative to antibiotics for broiler chickens. Further studies will focus on the development of the most effective delivery strategy for the B. subtilis-cNK-2 with scaling up technology to effectively apply this technology in commercial poultry production.

DISCLOSURES

SSW, IP, YL, CGG, and HSL declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. JGVO, LMR and CP are stockholders and employees of US Biologic.

ACKNOWLEDGMENTS

This research was supported by USDA/NIFA SAS grant 2020-69012-31823 ; accession number 12023193. The authors would like to acknowledge the contribution of Dr. Hasan Nur, Dr. Sunhee Hong, and Mr. Michael Netherland Jr. to the gut microbiome analysis.

Data Availability Statement: The datasets generated for this study are available on request from the corresponding author.

Ethics Statement: The animal use and experimental protocols were reviewed and approved by the Beltsville Agricultural Research Center Small Animal Care Committee (Animal Protocol No. 20-002).

Author Contributions: HL, JGVO, IP, and CG designed the research. SSW, IP, and YL conducted the research. JGVO and LMR produced the Bacillus subtilis cNK-2. SSW and IP analyzed data. SSW drafted the manuscript. HL, JGVO, and CP reviewed and edited the manuscript to its final version. SSW, IP, YL, CG, and HL had responsibility for the content. All authors read and approved the final manuscript.

1 Presented in part at the 2023 Poultry Science Association Annual Meeting, July 10-13, 2023 and 2024 Poultry Science Association Annual Meeting, July 15-18, 2024.
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Further Reading

Abd El-Hack M.E. El-Saadony M.T. Saad A.M. Salem H.M. Ashry N.M. A.Ghanima M.M. Shukry M. Swelum A.A. Taha A.E. El-Tahan A.M. AbuQamar S.F. Essential oils and their nanoemulsions as green alternatives to antibiotics in poultry nutrition: a comprehensive review Poult. Sci. 101 2021 101584 34942519
