1. Introduction
Coccidiosis is a severe enteric disease in poultry caused mainly by protozoa from the
Eimeria genus. Coccidiosis costs the poultry industry about 3 billion US dollars annually worldwide due to high mortality, impaired growth, and high medical costs [
1]. Anti-coccidial drugs have been widely used to control coccidiosis, but drug resistance of
Eimeria species in chickens has become prevalent worldwide. Because of the disadvantages of current anti-coccidial drugs and vaccines [
2], alternative strategies to prevent coccidiosis in broilers are an urgent and unmet need in the poultry industry. Several natural products, such as medicinal plants, herbal extracts, essential oils, organic acids, and probiotics, have been reported to have beneficial effects for the prevention of coccidiosis [
3,
4,
5,
6,
7,
8].
Bacillus licheniformis, a Gram-positive endospore-forming probiotic, has either been observed or isolated from the digestive tract of broilers [
6]. Dietary supplementation with
B. licheniformis ameliorates the growth performance and relieves necrotic enteritis caused by
Clostridium perfringens in broilers [
9,
10,
11]. However, the beneficial effects of
B. licheniformis as probiotics in broilers infected with coccidian parasites are still limited. Only one study reported that
B. licheniformis can improve body weight gain, intestinal lesion score, and fecal oocysts in broilers challenged with mixed coccidia infection [
12]. The underlying mechanisms of how
B. licheniformis prevents coccidiosis of broilers are still unclear.
The commercial feed additive of
B. licheniformis is mainly produced by liquid-state fermentation and fermented products (
B. licheniformis spores only, without functional metabolites) are then directly mixed into the diet. Compared with liquid-state fermentation, our previous study demonstrated that
B. licheniformis can be produced by solid-state fermentation. Fermented products contain
B. licheniformis spores and functional metabolites (antimicrobial cyclic lipopeptide) [
13]. Furthermore,
B. licheniformis-fermented products also display antimicrobial activity against
C. perfringens and
Brachyspira hyodysenteriae in vitro [
13,
14]. Dietary supplementation with fermented products produced by
B. licheniformis can ameliorate growth performance, alleviate necrotic lesions, and improve intestinal morphology in broilers exposed to
C. perfringens challenge [
15,
16].
In addition, fermented products also modulate intestinal bacterial composition in broilers [
15]. It has been reported that
Eimeria tenella infection induces perturbation of the cecal microbiota in different strains of broilers by elevating pathogenic bacteria abundance and reducing beneficial bacteria abundance [
17,
18]. Therefore, based on our previous studies, we hypothesize that fermented products might alleviate coccidiosis by normalizing the cecal microbiota composition of broilers.
The specific objectives of the present study were to investigate the effects of fermented products produced by B. licheniformis on growth performance and cecal microbial community in broilers exposed to coccidial challenge. The findings provide a basis for the use of B. licheniformis-fermented products as a possible method for preventing coccidia in poultry.
2. Materials and Methods
The Institutional Animal Care and Use Committee of National Ilan University reviewed and approved the animal protocol for the current study (IACUC, protocol number 107-12).
2.1. Experimental Design
The fermented products were produced in our previous study and the concentration of
B. licheniformis and
B. licheniformis-derived antimicrobial cyclic lipopeptide (surfactin) in fermented products were 5 × 10
11 CFU/g and 10 mg/g, respectively [
16]. A total of 108 one-day-old male broiler chicks (Ross 308) obtained from a local commercial hatchery with an average body weight of 43.96 ± 0.05 g were randomly allotted to one of three treatments in a completely randomized design. Each treatment was distributed into six replicate cages with six birds each. Broilers were reared in stainless-steel, temperature-controlled cages (190 cm × 50 cm × 35 cm).
The treatments consisted of (1) basal diet without treatment (NC), (2) basal diet plus coccidial challenge (PC), and (3) basal diet plus the coccidial challenge and 1 g/kg of fermented products (FP). The experimental diets were formulated to meet or exceed the requirements of birds according to National Research Council recommendations (Nutrient Requirements for Poultry, 1994,
Table 1). In the FP group, the soybean meal in the basal diet was replaced with fermented products equally. No coccidiostats and antibiotics were included in the diets. The broilers were on the test diets from 1 to 35 days of age. The feeding program had two phases that spanned days 1–20 and days 21–35.
The birds were given drinking water and feed ad libitum. A 23 light: 1 dark photoperiod was applied for the first week and then a 20 light: 4 dark photoperiod was applied after the first week. Ambient temperature on days 1 to 3 was set at 33 °C and gradually reduced to 30 °C on days 4 to 7, 27 °C on days 8 to 14, and 24 °C on days 15 to 35.
Newcastle disease–infectious bronchitis vaccination programs were performed on days 4 and 14 by nose drop administration (multivalent vaccine containing live Newcastle disease virus, B1 type, B1 strain and live IB virus, Massachusetts and Connecticut serotypes, Zoetis, Parsippany, NJ, USA). The commercial coccidial vaccine (Coccidiosis Quadrivalent Vaccine for Chickens, Guangdong Wens Dahuanong Biotechnology, Guangdong, China) containing anti-coccidial-sensitive strains of E. tenella, E. maxima, E. necatrix, and E. acervulina is a live oocyst isolated from chickens. A dose of the vaccine (1×), as recommended by the manufacturer, contains approximately 1100 oocysts. To mimic the Eimeria species challenge, a 10× dose of the commercial coccidial vaccine (approximately 11,000 oocysts and dissolved in distilled water) was administered to broilers in challenged groups (PC and FP) by oral gavage on day 14, whereas birds in the unchallenged group (NC) were orally gavaged with distilled water. Body weight and feed intake on a pen basis were evaluated every week and every day, respectively. The growth performance (average body weight, average daily gain, average daily feed intake, and feed conversion ratio) was calculated from two feeding phases (days 1–20 and days 21–35). The mortality of broilers was monitored daily.
2.2. Evaluation of Anti-Coccidial Index
The anti-coccidial index (ACI) was calculated based on the following formula, ACI = [relative body weight gain (RBWG, %) + survival rate (SR, %)] − [lesion score index (LSI) + oocyst count index (OI)]. The RBWG and SR of all broilers were recorded from days 14 to 35. For LSI analysis, two broilers per replicate were selected at the end of the experiment (day 35) based on their cage’s average body weight and then euthanized using carbon dioxide inhalation. Both ceca from each broiler were freshly collected for macroscopic LS evaluation using the method established by a previous study [
19]. Two broilers per replicate were chosen based on their cage’s average weight for OI analysis. Feces from each broiler were freshly collected daily from days 14 to 35 in an independent cage. After daily fecal sample collection, fresh feces from two broilers were weighed, pooled, suspended in water, and counted on McMaster egg-counting chambers (Vetlab Supplies, West Sussex, United Kingdom). Oocysts per gram of feces (OPG) were calculated from the average of 3 counts of each fecal sample. OI was calculated as follows: 100 × 0.4 × (oocyst counts per group)/oocyst counts for the coccidial challenge alone group.
2.3. 16S Ribosomal RNA Gene Sequencing and Data Processing
For microbiota analysis, two broilers per replicate were chosen at the end of the experiment (day 35) based on their cage’s average weight and then euthanized using carbon dioxide inhalation (birds chosen for microbiota analysis were identical to those for LSI analysis). Fresh digesta from the cecum of two broilers were sampled and pooled from each replicate. Three replicates (n = 3) were used for 16S ribosomal RNA gene sequencing. The total genomic DNA from cecal digesta was extracted and purified using a ZymoBIOMICS DNA Miniprep kit (Zymo Research, Irvine, CA, USA). Total DNA quantitative and qualitative analyses were measured by a Quantus Fluorometer (Promega, Madison, WI, USA) and agarose gel electrophoresis, respectively. The V3–V4 hypervariable region of the 16S rRNA gene from individual samples was amplified using 341F-805R primer (5′-CCTACGGGNGGCWGCAG-3′ and 5′-GACTACHVGGGTATCTAATCC-3′). The PCR amplicons were purified using a QIAquick Gel Extraction kit (QIAGEN, Germantown, MD, USA). Sequencing libraries were produced and sequenced at a read length of 300 nucleotides on a MiSeq platform (Illumina, San Diego, CA, USA). The sequence data were processed using the QIIME 2 software package (version 2017.4, GitHub, San Francisco, CA). High-quality reads were selected and all of the effective reads from all samples were clustered into operational taxonomic units (OTUs) based on 97% sequence similarity using UCHIME (version 4.2, GitHub) and mothur software (version 1.39.5, GitHub). Alpha diversity (richness and evenness) and phylogenetic assignment were accessed using QIIME 2 software (version 2017.4, GitHub) and naïve Bayesian classification method, respectively. The principal component analysis (PCA) and principal coordinate analysis (PCoA) based on the unweighted and weighted UniFrac distance matrices were used to visualize the difference of microbiota among groups using the R packages (version 3.5.0 and version 1.7.13, GitHub). The functions of all the OTUs were predicted by Kyoto Encyclopedia of Genes and Genomes (KEGG) databases using PICRUSt software (version 1.1.4, GitHub). Correlation analysis was performed using the Correlogram (version 0.84, GitHub).
2.4. Cecal Short-Chain Fatty Acid Measurement
For cecal short-chain fatty acid extraction, two broilers per replicate were chosen at the end of the experiment (day 35) based on their cage’s average weight and then euthanized using carbon dioxide inhalation (birds chosen for short-chain fatty acid analysis were identical to those for LSI and microbiota analysis). Fresh digesta from the cecum of two broilers were sampled and pooled from each replicate. Three replicates (n = 3) were used for the quantification of short-chain fatty acid. The short-chain fatty acids were analyzed by gas chromatography-mass spectrometry (Bruker GC-MS System, Burker Corp., Billerica, MA, USA). Briefly, cecal digesta was extracted with 10% isobutanol and homogenized. After centrifugation, the supernatant was isolated and mixed with NaOH and chloroform. The aqueous phase of the mixture was mixed with isobutanol, pyridine, and isobutyl chloroformate and sonicated. The mixture was then extracted with hexane. After centrifugation, the short-chain fatty acid contents in the supernatant were analyzed by gas chromatography-mass spectrometry. The separations were performed on low-bleed GC/MS columns (VF-5ms, 30 m × 0.25 mm; Agilent, Santa Clara, CA, USA) at a flow rate of 1.0 mL/min helium as a carrier gas. The electron energy was 70 eV. The oven temperature was held at 40 °C for 5 min, then ramped to 310 °C at a rate of 10 °C min−1. Injection volumes for all samples and standards were 2.0 L with a 10:1 split ratio. The cecal short-chain fatty acids measured were formic, acetic, propionic, butyric, and isobutyric acid.
2.5. Statistical Analysis
Replicates were considered to be the experimental units. Individual cages were defined as replicates for each determined parameter. The Student’s t-test (two-tailed) was used for intergroup comparison in SAS software (version 9.4, 2012; SAS Institute, Cary, NC, USA). p ≤ 0.05 was considered statistically significant. The PCoA analysis based on UniFrac distances coupled with standard multivariate statistics was assessed. The relationship between the dominant 10 genera, growth performance, and short-chain fatty acid levels was assessed using Pearson’s correlation coefficient (r).
4. Discussion
The overuse of drugs for coccidiosis in poultry leads to anti-coccidial drug resistance in parasites. Hence, probiotics have been considered as alternative candidates for anti-coccidial drugs. It has been demonstrated that
B. licheniformis can ameliorate body weight gain, intestinal lesion score, and fecal oocysts in broilers challenged with mixed coccidia infection [
12]. Our previous study demonstrated that fermented products produced by
B. licheniformis containing antimicrobial lipopeptides had similar benefits as antibiotics in the growth performance of broilers [
15]. We further confirmed that fermented products produced by
B. licheniformis exhibited anti-coccidial activity in broilers in the present study. In addition, fermented products could modify the cecal microbial community by increasing the genus
Lactobacillus abundance and decreasing the genus
Ruminococcus_torques_group abundance. Similar to a previous study [
15], the abundance of the genres
Lactobacillus and
Ruminococcus_torques_group were also positively and negatively correlated with the growth performance of broilers, respectively. The main findings of this study suggest that fermented products produced by
B. licheniformis can normalize
Eimeria species-induced adverse impacts on average weight gain and cecal microbiota of broilers.
Gut microbial balance plays a critical role in maintaining the health and growth of poultry by modulation of the nutrient digestion, intestinal function, and immune system [
20]. The intestinal microbiome can be affected by host and diet and overgrowth of pathogenic bacteria in the gut leads to systemic infection [
20]. It has been reported that
E. tenella infection can cause an intestinal microbial imbalance in broilers by increasing the pathogenic bacteria abundance and decreasing the beneficial bacteria abundance, thereby promoting gut damage [
17,
18]. The
Ruminococcus torques group genus is associated with gastrointestinal diseases by the degradation of mucin in the gastrointestinal tract, resulting in facilitating gut dysfunction [
21,
22].
In broilers, the genus
Ruminococcus torques group abundance is inversely correlated with the growth performance [
15,
23]. Thus, the genus
Ruminococcus torques group can be considered as pathogenic bacteria. In the present study, fermented products produced by
B. licheniformis can normalize the genus
Ruminococcus torques group abundance in the cecum of broilers under coccidial challenge. The genus
Ruminococcus torques group abundance is negatively associated with the growth performance in broilers under coccidial challenge, which is in agreement with previous studies [
15,
23]. In beneficial bacteria, it has been demonstrated that
Lactobacillus species are able to inhibit
E. tenella sporozoite invasion in vitro [
24].
Lactobacillus-based probiotics also exhibit anti-coccidial properties in broilers [
2,
25]. In the present study, fermented products can increase the genus
Lactobacillus abundance in the cecum of broilers exposed to coccidial challenge. Furthermore, the genus
Lactobacillus abundance is positively associated with the growth performance in broilers under coccidial challenge, which is also in agreement with previous studies [
15,
23]. These results imply that fermented products produced by
B. licheniformis may inhibit
Eimeria oocyst development in the cecum of broilers by increasing the genus
Lactobacillus abundance and decreasing the genus
Ruminococcus torques group abundance. In our study, some bacteria are specifically sensitive to fermented products or coccidial challenge treatment in the cecum of broilers, such as genus
Romboutsia. It has been reported that the administration of
Lactobacillus species can decrease the genus
Romboutsia abundance in the feces of laying hens and also improve the laying rate [
26]. In the present study, the genus
Romboutsia abundance is negatively correlated with the genus
Lactobacillus abundance in broilers, which is in agreement with a previous study [
26].
We also observed that fermented products improve the average daily gain and also decrease the abundance of the genus
Romboutsia in the cecum. Thus, these findings imply that fermented products specifically attenuate the genus
Romboutsia abundance and the genus
Romboutsia may play a significant factor in the growth traits of poultry. In addition to the genus
Romboutsia, the genera
Lachnospiraceae_unclassified and
Sellimonas was specifically decreased in broilers exposed to coccidial challenge (PC and FP group). The genus
Lachnospiraceae_unclassified may have a beneficial effect on gut development and health by the production of short-chain fatty acids [
27]. A recent study has demonstrated that the genus
Sellimonas is reduced in abundance in the hens challenged with
Salmonella Typhimurium [
28]. However, the abundance of the genera
Lachnospiraceae_unclassified and
Sellimonas in the cecum of broilers were not affected by fermented products in our study. Therefore, the role of genera
Lachnospiraceae_unclassified and
Sellimonas in the cecum of broilers still need to be confirmed. Taken together, these results suggest that fermented products increase certain beneficial bacteria populations and reduce the pathogenic bacteria populations in the gut of broilers. The modification of gut microbiota by fermented products can help to prevent coccidiosis in broilers.
In the cecum, the short-chain fatty acid and microbial community exert an important role in maintaining gut health and promoting growth in broilers by regulating the intestinal morphology and immune response [
29]. Short-chain fatty acids and microbial communities interact with each other via a complicated mechanism in order to create a beneficial environment for the growth of broilers [
30]. In this study, some bacteria (
Sellimonas,
Romboutsia, and
Ruminococcus torques group) in the cecal digesta were inversely correlated with short-chain fatty acid levels, indicating that these short-chain fatty acids in the cecum might inhibit these bacteria growth. These bacteria were also negatively associated with the growth performance (BW, ADG, and ADFI), implying that these bacteria might be unfavorable to gut health. The genus
Lactobacillus abundance was positively associated with the short-chain fatty acid levels in the cecum and growth performance (BW and ADG), indicating
Lactobacillus could prevent coccidiosis and improve growth by production of short-chain fatty acids. A strongly negative correlation between the genre
Lactobacillus and
Ruminococcus torques group was also observed in the present study. The administration of
Lactobacillus in the diet can improve intestinal health and reduce the mortality of broilers suffering from necrotic enteritis [
31]. To sum up,
Lactobacillus may inhibit the growth of harmful microbes in the cecum by the production of short-chain fatty acids, thereby improving the gut health and growth in broilers under coccidial challenge.
The antimicrobial mechanisms of antimicrobial lipopeptide have been widely proposed [
32]. Previous studies have reported that
B. licheniformis can synthesize antimicrobial lipopeptides [
33,
34]. Surfactin, one of
B. licheniformis-derived antimicrobial lipopeptides, exhibits antibacterial activity against a wide range of Gram-positive bacteria, such as
Listeria monocytogenes and Methicillin-resistant
S. aureus, but does not cause hemolysis [
35,
36]. Our previous findings have demonstrated that surfactin isolated from fermented products inhibits the growth of
C. perfringens and
B. hyodysenteriae [
13,
14]. In addition to bacteria, surfactin also exhibits anti-parasitic activity against
Nosema ceranae and
Plasmodium falciparum [
37,
38]. Surfactin can reduce parasitosis development of
N. ceranae by direct exposure to spores of
N. ceranae, resulting in a reduction in infectivity [
38] Surfactin is also an inhibitor of intraerythrocytic growth of
P. falciparum through inhibition of NAD+ and acetylated peptide [
37].
Our preliminary results (data not shown) have demonstrated that surfactin purified from
B. licheniformis-fermented products inhibits sporulation of the
Eimeria oocyst and promotes the death of merozoite in vitro, implying that surfactin may attack
Eimeria species directly. In addition to antimicrobial activity, surfactin also exhibits an inhibitory effect on lipopolysaccharides-induced inflammation in vitro [
39]. Moreover,
B. licheniformis also normalizes the gut microbiota, thereby creating a healthy gut environment by competitive exclusion of pathogens for the prevention of
Eimeria species infection. Thus, the potential anti-coccidial mechanisms we proposed are (1) surfactin inhibits
Eimeria oocyst growth in the gut, (2) surfactin promotes immunomodulation in the gut mucosal immune system, and (3)
B. licheniformis regulates microbial community by competitive exclusion of pathogens or production of antimicrobial lipopeptides. However, the precise mechanism of how fermented products exert anti-coccidial activity in the prevention of coccidiosis remains to be investigated.
Although the diets were formulated to meet or exceed the requirements of Ross 308 broiler, broilers were kept in the cage in the present study and body weight was less than expected at 35 days of age (–7.6%) compared with the Ross 308 broiler management guide 2019 [
40]. Aviagen management handbook is mainly designed to optimize the growth of Ross 308 broiler for commercial purposes using a floor litter rearing system with advanced environmental control. Previous studies have reported that the body weight of broilers from the floor litter group is heavier (+9.6 to 12.0%) than those from the cage group [
41,
42,
43]. Therefore, the difference between rearing systems is a possible reason why the lower body weight of broilers was observed in the present study. It has been demonstrated that coccidial challenge without vaccination has lower body weight and higher feed conversion ratio in broilers [
44]. In our study, the body weight (35 days of age), average daily gain (day 21 to 35 and day 1 to 35 of age), and feed conversion ratio (day 21 to 35 and day 1 to 35 of age) are worsened in the PC group, which is in agreement with a previous study [
44]. Fermented product supplementation could normalize the body weight (35 days of age) and average daily gain (day 21 to 35 and day 1 to 35 of age) in broilers exposed to coccidial challenge. However, the feed conversion ratio of the FP group was not improved at 21 to 35 days and 1 to 35 days of age since the average daily feed intake was increased. The increased feed intake was also observed in the PC group. There is no clear explanation for the increased feed intake in the PC and FP group, but we can speculate that the nutrient digestion and absorption in the PC group is severely impaired due to coccidial challenge compared with the FP group. It has been reported that the blood glucose levels of broilers under coccidial challenge are decreased, and blood glucose levels are negatively associated with appetite [
45,
46]. Thus, the blood level in the PC group is supposedly lower due to the low efficiency of nutrient absorption, thereby stimulating appetite in the hypothalamus. fermented products may promote nutrient utilization by the production of digestive enzymes, resulting in an increased weight gain. In addition, it has been reported that probiotics (such as
Lactobacillus) can stimulate appetite by activation of ghrelin receptor (hunger signal) in the hypothalamus [
47]. Therefore, the increased feed intake in the PC and FP groups may be regulated by a different mechanism.
The cellular community–prokaryotes of KEGG function includes quorum sensing and biofilm formation [
48]. Bacteria can regulate the virulence factor production by quorum sensing and virulence factors increase pathogen colonization, immunoevasion, and immunosuppression [
48]. The bacterial biofilm formation promotes pathogen adhesion to the surface of the gut and it also associates with antimicrobial resistance [
49]. In our study, the cellular community–prokaryotes function was up-regulated in the PC group, indicating that
Eimeria species may stimulate pathogen colonization in the gut and suppress host immune response. However, the effect of cellular community–prokaryotes function in the cecum of broilers was decreased by fermented products in broilers exposed to coccidial challenge. The results also support the potential mechanism that fermented product supplementation may create a healthy gut environment by competitive exclusion of pathogens. Furthermore, the anti-coccidial index evaluated by relative body weight gain, survival rate, lesion score index, and oocyst count index was worsened in the PC group, whereas fermented products produced by
B. licheniformis could increase the anti-coccidial index of broilers under coccidial challenge. This finding is in agreement with the results of Chaudhari et al. [
12], who observed that
B. licheniformis can improve body weight gain, intestinal lesion score, and fecal oocysts in broilers challenged with mixed coccidia infection. Taken together, fermented products produced by
B. licheniformis can regulate microbial community by competitive exclusion of pathogens for the prevention of
Eimeria species infection in broilers.