Supplemental illite or in combination with probiotics improves performance and intestinal health in broiler chickens challenged with Salmonella typhimurium

The experimental protocol was approved (CBNUA-2148-23-01) by the Institutional Animal Care and Use Committee of Chungbuk National University, Cheongju, Korea.

The chemical composition of illite, which provided by Garam (Eumseong, Korea), is shown in Table 1. In this study, 1 × 10⁸ CFU/kg of C. butyricum and B. subtilis were used (Garam, Eumseong, Korea). The ST was provided in stock form. The ST was thawed, and ten microliters were mixed with 10 mL of nutrient broth, cultivated at 37°C for 24 h, and then sub-cultured at approximately 1.0 × 10₇ CFU/mL.

A total of 72 one-day-old Arbor Acres broilers were randomly assigned to three groups based on their initial body weight (BW) of 35.28 ± 0.34 g, with six replicate cages (W: 173 cm, D: 63 cm, H: 55 cm) per group and three birds per replicate. The experimental period lasted for 28 d. Dietary treatments included the following: 1) NC, non-challenge control, birds fed with basal diet; 2) CC, ST challenge control, birds fed with basal diet; 3) IA, the CC with 1% illite alone (10 g/kg); 4) ICB, the IA with 0.1% CB (1 × 10⁸ CFU/kg). The experiment initiation temperature was 31 ± 1°C, and then the temperature was gradually lowered to maintain 22 ± 1°C. All broilers except NC group were orally inoculated with a total of 1.5 mL and 2.1 mL ST (1 × 10⁷ CFU/mL) for 3 consecutive days on 8 and 15 d, respectively. All diets were formulated to meet or exceed the nutrient requirements for poultry by the NRC [36]. Compositions of basal diets are shown in Table 2. Broilers were fed ad libitum diet and water.

At 7, 14, 21, and 28 d, all broilers and remaining diet in the cages were weighed at each time point to determine the BW, BWG, feed intake (FI), and FCR. The BWG was calculated as the BW of the previous time point was subtracted from the BW of the current time point. The FI was calculated by subtracting the remaining diet amount from the initial diet amount, and FCR was calculated by dividing FI by BWG.

Broilers were fed diets mixed with 0.2% chromium oxide (Cr₂O₃) for 3 consecutive days from 11 d and 25 d, and fecal samples were collected during that period. At the same time, diet samples were collected. After collection, fecal and diet samples were stored in a freezer at −20°C, immediately. At the end of the experiment, fecal samples were dried at 70°C for 72 h and then crushed on a 1 mm screen. The procedures utilized for the determination of dry matter (DM) and crude protein (CP) digestibility were conducted with the methods by the AOAC [37]. The gross energy (GE) of the diets and feces was analyzed by using an adiabatic oxygen bomb calorimeter (Parr 6400, Parr Instruments, Moline, IL, USA). Chromium levels were determined via UV absorption spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan) using the Williams et al. [38] method. The apparent total tract digestibility (ATTD) percentage was calculated using the following equation:

The fecal scores were individually recorded at 08:00 and 17:00 by the same person during the entire experimental period. The fecal score was scored using a method used by Cooper et al. [39]. The fecal score was as follows: 0, normal dropping; 1, normal to pasty; 2, liquid; 3, liquid with blood; 4, bloody droppings.

At 14 and 28 d, blood samples (2 mL each) were collected from the brachial wing vein into a sterile syringe. At the time of collection, blood samples were collected in a vacuum tube containing K₃EDTA for complete blood count analysis and nonheparinized tubes for serum analysis, respectively. After collection, blood samples were centrifuged at 12,500×g at 4°C for 20 min. Red blood cell, white blood cell, heterophil, and lymphocyte were analyzed with a hematology analyzer (XE2100D, Sysmex, Kobe, Japan). interleukin (IL)-6 and tumor necrosis factor α (TNF-α) concentrations were determined using commercially available ELISA kits (Quantikine, R&D Systems, Minneapolis, MN, USA), and the absorbance was measured at 450 nm.

At 14 and 28 d, fecal samples were collected in conical tubes. Fecal samples were stored on ice and analyzed immediately. From the sample, 0.1 g was suspended in 1 × phosphate buffered saline (PBS; GenDEPOT, Katy, TX, USA), homogenized, and diluted from 10⁻⁴ to 10⁻⁷ to count the number of bacteria. Evenly spread 100 µL of the diluted solution on the agar. Brilliant Green (BG) Sulfa agar (KisanBio, Seoul, Korea) was used for Salmonella, and De Man–Rogosa–Sharpe (MRS) agar (KisanBio) was used for Lactobacillus. Salmonella was cultured for 24 h 37°C, and Lactobacillus was cultured for 48 h 37°C. Immediately after removal from the incubator, Salmonella and Lactobacillus were counted, and statistical analysis was performed by converting them to logs.

Six broilers per treatment were sacrificed at the end of the experiment to collect ileal tissue samples. The tissue sample for morphological measurements was taken from the ileal segment (2 cm anterior to the ileocecal valve), rinsed clean with 10% neutral buffered formalin (NBF; Sigma-Aldrich, St. Louis, MO, USA). The intestinal segment was submerged in approximately 20 mL of 10% NBF for 24 h. Slides of intestinal cross-sections (5 μm thick) were treated with paraffin and stained with hematoxylin and eosin. The slides were examined using an inverted phase-contrast microscope (Olympus IX51, Olympus Corporation, Tokyo, Japan). The villus height (VH) was measured from the tip of the villus to the crypt orifice. The crypt depth (CD) was measured from the junction of the villus to the crypt base. And then, the VH to CD ratio (VH:CD) was calculated.

All data except for frequency of diarrhea were analyzed by one-way ANOVA using JMP (JMP Pro version 16.0.0, SAS Institute, Cary, NC, USA), using each pen as the experimental unit. The results are presented as the mean ± standard error of the mean. Differences between treatment means were determined using Tukey’s multiple range test. A probability level of p < 0.05 was indicated to be statistically significant, and a level of 0.05 ≤ p < 0.10 was considered to have such a tendency. The frequency of diarrhea was analyzed contingency analysis to test the relationship between categorical variables (scores) and the different combinations tested in this study. A Chi-square test was performed to determine if the different combinations had an effect on the categorical variables repartition with significance accepted at p ≤ 0.05, and visualized using GraphPad Prism 9.5.1 (GraphPad, San Diego, CA, USA).

The CC group showed lower (p < 0.05) BW compared with the NC group at 21 d (Table 3). Additionally, during the 1st challenge period and over the entire period, the CC group showed a higher (p < 0.05) FCR compared to the NC group. While the ICB group showed increased (p < 0.05) BW compared with the CC group at 21 d. Also, compared with CC group, there was a higher (p < 0.05) BWG and a lower (p < 0.05) FCR in broilers fed IA and ICB at 15 to 21 d. During the entire experimental period, the IA group and ICB group showed a higher tendency (p = 0.065) for BWG and lower (p < 0.05) FCR than the CC group.

At 14 and 28 d, the CC group and IA group showed lower (p < 0.05) ATTD of DM than the NC group (Table 4). However, group of broilers fed diets with ICB increased (p < 0.05) ATTD of DM compared with CC group at 28 d.

These observed fecal score was statistically different among the four dietary treatments (p < 0.05; Fig. 1). The CC group showed the highest score 2 (35.71%), which is considered as diarrhea, and ICB group showed a lower level at 12.8% compared to the CC group.

Fig. 1. Effects of illite and probiotics supplementation on fecal score of Salmonella enterica serotype typhimurium-challenged broilers Score 0, normal dropping; 1, normal to pasty; 2, liquid; 3, liquid with blood; 4, bloody droppings. χ²= 147.679, p < 0.001. NC, non-challenge control, birds fed with basal diet; CC, Salmonella enterica serotype typhimurium challenge control, birds fed with basal diet; IA, CC with 1% illite alone; ICB, IA with 0.1% of Bacillus subtilis (1 × 10⁸ CFU/kg) and Clostridium butyricum (1 × 10⁸ CFU/kg), respectively.

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At 14 d, the CC group had higher (p < 0.05) heterophil and TNF-α than the NC group (Table 5). Additionally, the CC group was significantly higher (p < 0.05) IL-6 and TNF-α than the NC group at 28 d. On the other hand, the IA group and ICB group showed no significant differences (p > 0.05) in TNF-α, and IL-6 compared with the NC group.

At 14 and 28 d, the CC group showed a higher (p < 0.05) Salmonella count and lower (p < 0.05) Lactobacillus count in feces than the NC group (Table 6). However, the ICB group showed a lower (p < 0.05) Salmonella count in feces than the CC group at 14 and 28 d. Also, at 28 d, the IA group and ICB group had a significantly higher (p < 0.05) Lactobacillus count in feces than the CC group.

Compared with the NC group, the CC group showed decreased (p < 0.05) VH and VH:CD and increased (p < 0.05) CD (Table 7). However, the IA group and ICB group had no significant difference (p > 0.05) on the CD and VH:CD compared to the NC group. Moreover, the IA group and ICB group showed a lower (p < 0.05) CD and a higher (p < 0.05) VH:CD than the CC group.

Numerous studies have showed that ST challenges cause poor performance by inhibiting nutrient digestion, absorption [40–42]. Correspondingly, in our study, ST challenge decreased ATTD of DM, thereby resulting the impaired performance. Consistent with our study, Alkhulaifi et al. [7] has demonstrated that ST challenge exhibited an 11% decrease of ADG and a 9% increase of FCR compared to the non-challenged group in broilers at 11 to 25 d. Moreover, Shao et al. [43] has reported that ST challenge caused 7% reduction of BWG in broilers. However, in our study, supplementation with IA and ICB to diet in ST-challenged broilers improved FCR during the entire period, with no significant difference in the non-challenged group. This alleviation might be attribute to the antimicrobial properties of illite and CB. The Al₂O₃ and Fe₂O₃ are key elements for their antimicrobial efficacy, which is a main component of illite [44]. Also, previous study has reported that B. subtilis exerts beneficial effects on performance in weaning pigs through the production of antimicrobials [45]. Additionally, Zhang et al. [46] have found that C. butyricum produced substances that suppress pathogens, decreasing E. coli count in the cecal of broilers challenged with E. coli at 21 d. Similarly, the antimicrobial effect of ICB reduced the count of Salmonella in feces on 14 and 28 d. Also, IA and ICB have decreased the frequency of diarrhea by 13.83% and 22.91%, respectively. Diarrhea occurs due to disruption of the intestinal acid-base equilibrium balance caused by ST, and a decrease in the frequency of diarrhea indicates alleviated ST infection [47,48]. Therefore, our result revealed that dietary IA and ICB improved growth performance in ST-challenged broilers by suppressing Salmonella infection. However, contrary to our findings, previous research has shown that supplementation with C. butyricum and B. subtilis did not impact the performance of broilers challenged with Salmonella [49,50]. This reason could be attributed to differences in the time point of the challenge, animal model, as well as the dosages of Salmonella and probiotics used.

The digestion and absorption of dietary nutrients primarily occur in the small intestine [51]. The improvement of feed efficiency in broilers could partly explained by enhanced VH which leads to increase the capacity for absorbing nutrients [52,53]. Several studies have revealed that adding illite improved VH and nutrient digestibility (such as DM, CP, and GE) in broilers and pigs [54–56]. Nevertheless, our study did not show the effects of illite supplementation on the VH as well as nutrition digestibility in broilers. However, inconsistent with IA, supplementation with ICB to diet significantly increased the ATTD of DM at 28 d, possibly due to the complementary effect of illite and probiotics. According to the previous studies, Zhang et al. [32] and Mohamed et al. [57] have reported that B. subtilis and C. butyricum enhance digestion by boosting the activity of enzymes in the gastrointestinal tract and might be involved in improving digestion and absorption. Silicates also produce sticky mucus, which slow down the transit time of digesta [58]. This effect might be further amplified when combined with the enzyme activity of probiotics. The exact mechanism of increased nutrition digestion by the synergy effect has not been documented. However, we speculate that an elevated ATTD of DM could be attributed to distinct mechanisms of illite and probiotics.

The ST induces intestinal inflammation through the production of enterotoxins and compromises the integrity of the intestinal epithelium, leading to villus atrophy [59,60]. Thereby, enterocyte proliferation occurs in the crypts, resulting in a deeper crypt [61,62]. In brief, deeper CD suggests the presence of harmful bacteria and toxins in the intestines. In the present study, ST challenge damaged intestinal mucosa, as observed by decreased VH and increased CD, which is consistent with previous studies [63,64]. However, dietary IA and ICB alleviate negative effects (including an increase in CD and a decrease in VH:CD) caused by ST, suggesting a reduction in intestinal Salmonella bacterial load and toxin activity. The Al³⁺ and Fe²⁺ cations present in the interlayer space structure of illite can primarily bind to the lipopolysaccharide molecules produced by gram-negative bacteria (such as ST), thereby contributing to the overall health of the gut [65,66]. Also, numerous studies have showed that supplementation with probiotics improves intestinal morphology by decreasing the number of Salmonella in the intestines [67–70]. Actually, in this study, the addition of IA and ICB reduced the counts of Salmonella in the feces, which can support the intestinal morphology findings of this study. Also, reduction in Salmonella count by ICB may emerge from a complementary effect of combining clay mineral and probiotics. Previously, Han et al. [71] has stated that the adsorption ability of clay mineral from lipopolysaccharides could more effectively help probiotic colonization in the intestines. This could be the reason why the combination of illite and CB was more effective than illite alone in reducing the Salmonella count in this study. Additionally, this mechanism may explain why supplementing with IA and ICB to diet in ST-challenged broilers has a higher count of Lactobacillus than the CC group in the feces at 28 d.

Pro-inflammatory cytokines are essential in initiating immune responses of the host [72,73]. However, their exaggerated or prolonged secretion may harm the host [74]. The increased levels of heterophils, IL-6, and TNF-α after the ST challenge suggest that the immune and inflammatory responses were overly activated [75–77]. Consistent with our results, Olfati et al. [78] and Milby-Blackledge et al. [79] have stated that ST challenge could lead to the release of pro-inflammatory cytokines in broilers. However, our study showed that both IA and ICB group led to a numerical reduction in the secretion of pro-inflammatory factors IL-6 and TNF-α compared with CC group in the serum. This result suggests that systemic inflammation was effectively reduced [80]. Consistently, previous studies have shown that silicate can reduce the activation of TNF-α and IL-6 by increasing antibody production and enhancing humoral immune function [81,82]. Also, our result is partially correlated with previous study’s evidence, suggesting that the dietary B. subtilis andC. butyricum reduce the TNF-α and IL-6 level in serum and liver, respectively, in Salmonella-challenged broilers [75,83]. According to [84,85], dietary B. subtilis and C. butyricum increased goblet cell production, inhibiting the binding of ST from epithelial cells, ultimately alleviating ST infection. However, other studies have showed that the supplementation with C. butyricum changed the immune sensitivity of broilers by increasing TNF-α and IL-6 mRNA expression, respectively [32]. Additionally, there are few studies about dietary illite supplementation on the cytokine level in animals infected with ST, thereby an exact mechanism is not presented for the anti-inflammatory effects of illite. This lack of mechanistic understanding hampers the ability to optimize dosages and combinations of these supplements for maximal efficacy. Also, hyperimmune of illite and CB may result in autoimmunity or conflicting interactions with the host immunity [86]. Therefore, our study suggested that additional research is needed on the mechanisms by which illite and CB supplementation affect the broiler immune system.