Inclusion of Quillaja Saponin Clarity Q Manages Growth Per-Formance, Immune Response, and Nutrient Transports of Broilers During Subclinical Necrotic Enteritis

1. Introduction

Clostridium perfringens alpha toxin (CPA) and the necrotic enteritis B like pore forming toxin (NetB) are produced by this anaerobic bacterium reported as the causative agent of necrotic enteritis (NE) in poultry [1,2,3]. NE is of economic importance to the industry as it has been estimated to cost approximately $6 billion in yearly losses [4,5]. To control pathogenic bacteria, sub-therapeutic doses of in-feed antibiotics have been used to improve broilers' welfare and overall productivity [6,7]. However, with consumer demands for chickens raised without antibiotics and legislative restrictions, the poultry industry is looking to raise more flocks without antibiotic growth promoters (AGPs) [8,9]. However, with the removal of AGPs, incidences of NE have increased, leading the industry to find alternative strategies to improve bird health during this enteric challenge [10,11].

One strategy currently being studied to reduce enteric diseases is nutritional interventions like phytogenic-based feed additives [12,13,14,15]. Phytogenic feed additives (PFA) are plant-derived bioactive compounds incorporated into animal feed or water [16,17,18]. They can improve productivity, available dietary energy, nutrient digestibility, innate immunity and host disease resistance, alter the gut microbiota, decrease the prevalence of pathogens, and prevent bacterial colonization of the gastrointestinal tract [15,19]. Quillaja saponaria (QS) is classified as a PFA that is both fat and water-soluble [20,21,22]. It is thought that the mechanism of action is their ability to penetrate the cell membrane affecting cellular permeability, which leads to leakage of critical molecules and ions from harmful bacteria [23,24,25]. However, there is still an ongoing need to alleviate the adverse effects caused by enteric diseases, as well as the need to better understand how these saponins improve performance and health. The central hypothesis was that Clarity Q would have a positive effect on birds’ performance and response during this subclinical NE model. Therefore, this study was conducted to determine the effects of the saponin based product Clarity Q on average daily gain (ADG), average daily feed intake (ADFI), feed conversion ratio (FCR), NE lesion scores, mRNA abundance of tight junction proteins, energy signaling pathways, and essential nutrient transport genes of broiler chickens during an NE challenge.

2. Materials and Methods

Day (d)-old Cobb male broiler chicks (n=1,200) were sourced from a local hatchery and randomly weighed and allocated to 4 treatment groups with each treatment consisting of 10 replicate pens and 30 birds per pen. Floor pens were ~1.22m × 2.44m and covered with fresh pine shavings as litter. The treatments included a negative control (NC) with birds fed a corn-soybean meal basal diet, a positive control (PC) fed the NC diet with bacitracin methylene disalicylate (BMD) at 50 g/metric ton (MT), and Clarity Q (CQ) added at 15 (CQ15) or 30 g/MT (CQ30). The diets were crumbled for the starter phase (d 0-14) and pelleted for the grower (d 15-28) and finisher (d 29-42) phases (Table 1). Chicks had access to feed and water ad libitum using a bucket-type feeder and a nipple drinker waterline. The light cycle was 24 h of light for the first three days, reduced to 23h: 1 h for d 4-7, and then reduced to 18 h of light and 6 h of dark for the remaining duration of the trial. An automatic ventilation system was used to control the environment, and the temperature was maintained as follows: 32°C for the first 3 days, then gradually reduced ~3°C each week until it reached 23°C at the start of week 4 where it remained constant. This study was conducted under the Institutional Animal Care and Use Committee guidelines.

2.1. Performance

Starting at placement, birds were monitored twice daily. Birds were weighed on d 8, 14, 28, and 42 on a per pen basis. If dead birds were found, date, body weight, and cause of death were recorded. This procedure continued throughout the 42 d trial to record mortality/treatment for each phase thus allowing for adjusting performance parameters such as body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), feed conversion ratio (FCR) and, adjustments for daily mortality.

2.2. Necrotic Enteritis Challenge and Lesion Scoring

As previously done in our lab, on day of placement, feed and litter were sprayed with 10 × coccidiosis vaccine (Advent^®; Huvepharma, Inc) containing live oocysts of Eimeria acervulina, E. maxima, and E. tenella [26,27,28]. When coupled with the presence of C. perfringens spores in the barn environment, this leads to the development of a NE outbreak around one week after vaccine application [26,27,28]. On d 8, based on average body weight of each pen, 3 birds were randomly selected and euthanized via cervical dislocation and the small intestine was removed to examine NE lesions. The duodenum and jejunum were scored separately based on a 0-4 scale system: 0 = no gross lesions, normal intestinal appearance; 1 = thin-walled or friable, gray appearance; 2 = thin-walled, focal necrosis, gray appearance, small amounts of gas production; 3 = thin-walled, sizable patches of necrosis, gas-filled intestine, small flecks of blood; 4 = severe extensive necrosis, marked hemorrhage, large amounts of gas in the intestine [29] by personnel blinded to the treatments.

2.3. Total RNA Extraction and Reverse Transcription

On d 8 and d 14, one bird from each pen) was humanely euthanized and ~2-cm sample immediately cut from the middle section of the jejunum, rinsed in cold PBS, snap-frozen in liquid nitrogen, and stored at −80℃ until analysis. The samples were homogenized by a bead mill homogenizer (TissueLyser II, Qiagen) and total RNA was extracted with Trizol reagent following the manufacturer's instructions (ZYMO Research, Direct-zol RNA Miniprep). Total RNA concentration was determined at optical density (OD) 260 (Nanodrop 1, Thermo Fisher), and RNA purity was verified by evaluating the ratio of OD 260 to OD 280. After extraction, 2 μg of total RNA were reverse-transcribed into cDNA using the high-capacity cDNA Reverse Transcription kit (Applied Biosystems) following the manufacturer's protocol, and the cDNA was stored at −20°C.

2.4. Quantitative Real-Time PCR

Quantitative real-time PCR (qRT-PCR) was performed using the QuantStudio 3 (Applied Biosystems) using PowerTrak Fast SYBR^TM Green Master Mix (Applied Biosystems). The cDNA was diluted 1:20 in nuclease-free water, and 1.5 μL of the diluted cDNA were added to each well of a 96-well plate. Next, 8.5 μL of real-time PCR master mix containing 5 μL of Fast SYBR Green Master Mix (Applied Biosystems), 0.5 μL each of forward and reverse primers (Table 2), and 2.5 μL of sterile nuclease-free water were added to each well for a final reaction volume of 10 μL. During the PCR reaction, samples were subjected to an initial denaturation phase at 95°C for 120 s followed by 40 cycles of denaturation at 95°C for 5 s and annealing and extension at 60°C for 30 s. Each target reaction was performed in duplicate wells. Product specificity was confirmed by analysis of the melting curves produced by the QuantStudio 3. mRNA abundances for tight junction proteins occludin (OCLN), claudin (CLDN) 1 and 5, zonula occludens (ZO) 1 and 2, and junctional adhesion molecules (JAM) 2 and 3, as well as the nutrient transporter genes excitatory amino acid transporter 3 (EAAT3), glucose transporter (GLUT) 2 and 5, peptide transporter (PepT) 1 and 2, and sodium glucose transporter (SGLT) 1 were analyzed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control. Cellular energy homeostasis pathways were also analyzed via AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), and peroxisome proliferator activated receptor gamma coactivator one alpha (PGC-1α), as well as mucin 2 (MUC2), pro-inflammatory gene tumor necrosis factor receptor associated factor 3 (TRAF3) and anti-inflammatory gene Annexin (ANXA1). Average mRNA abundance relative to GAPDH for each sample was calculated using the 2^−ΔΔCt method. The calibrator for each gene was the average ΔCt value from the negative control group of each corresponding day.

2.5. Statistical Analysis

All data were subjected to a one-way ANOVA using the JMP Pro 16 program, a Chi-squared test was used for lesion scores. The Fisher’s least significant difference (LSD) test compared separated means when significant differences were noted. Statistical differences were considered significant at P ≤ 0.05.

3. Results

3.1. Performance Parameters

Performance data are presented in Table 3. Body weight displayed no significant difference between treatments throughout the 42 d trial. However, both CQ15 and CQ30 had numerically higher weights on d 28 (1,381.27 and 1,389.33 g/bird, respectively) and 42 (3,165.79 and 3097.07 g/bird, respectively) compared to NC and PC. Average daily gain was similar for all treatments during the first two weeks. During d 0-28, the PC (55.59 g/bird) and CQ15 (55.81 g/bird) groups had significantly greater ADG compared to CQ30 (53.09 g/bird) but were comparable to NC. Average daily feed intake in CQ30 and NC were lower compared to CQ15, but comparable to PC during d 9-14 and d 0-14. Overall (d 0-42), CQ30 had a numerically lower ADFI and FCR compared to all other treatments.

3.2. Mortality Rate

Mortality rate is presented in Figure 1. There were no statistical differences amongst treatment, however both CQ supplemented treatments showed a reduction in percent mortality compared to NC and PC.

3.3. Necrotic Enteritis Lesion Scores

The effect of the dietary supplementation of the saponin-based product Clarity Q on necrotic lesions is presented in Figure 2. On d 8, although there were no statistical differences in lesion scores amongst treatments, CQ15 and PC supplements reduced NE lesions in the duodenum and jejunum, respectively, compared to all other treatments.

3.4. mRNA Abundance of Tight Junction Proteins, Cellular Energy Homeostasis Pathways, and Nutrient Transporters

Figure 3, Figure 4 and Figure 5 show the effects of dietary supplementation of the saponin-based product Clarity Q on mRNA abundance of genes for tight junction proteins, markers of cellular energy homeostasis, and nutrient transporters. On d 8 (peak infection), CQ30 birds exhibited a significantly greater abundance of CLDN1 and CLDN5 (P = 0.0016 and P = 0.0038 respectively) compared to NC and PC, as well as AMPK compared to all other treatments. Both CQ15 and CQ30 treatment groups exhibited greater abundance of EAAT3, GLUT2, and PepT2 compared to NC. GLUT5 mRNA abundance was greater in PC birds (P < 0.0001) compared to all treatments. On d 8 and d 14, abundance of MUC2 and PGC1-α was significantly lower in CQ15 and CQ30 compared to NC and PC. On d 14 (recovery phase), OCLDN, CLDN1, JAM2, and ZO1 showed a significantly lower abundance in CQ15 and CQ30 compared to NC and PC. However, mRNA abundance of ZO2 was significantly greater in CQ30 compared to all other groups. Annexin 1 was significantly reduced in CQ30 while that of PepT1 was lower compared to NC and PC on d 14; and PepT2 was greater in CQ15 and CQ30 compared to NC and PC.

4. Discussion

Subclinical NE inflicts one of the greatest economic impacts in poultry production mostly due to reduction in feed efficiency [30]. In the current study, CQ30 showed a reduction in ADFI on d 9-14 and d 0-14 and caused a slight decrease in ADFI during the overall experimental period. This contributed to the overall better FCR in the CQ30-supplemented group although these results were not statistically significant. Moreover, PC and CQ15 groups significantly increased ADG compared to CQ30 during d 0-28, showcasing that CQ supplementation could promote broiler performance similarly to an AGP. Similar studies have shown results supporting both positive and no effects of phytogenics on feed conversion under normal conditions or after a challenge with C. perfringens [31,32,33,34,35]. These varying effects on performance are not uncommon amongst studies and could be associated with the health of the flock, type of basal diet, and environmental conditions.

Damage to the intestinal mucosa is an important factor for C. perfringens colonization and the presence of a coccidial infection is the most common causative factor facilitating C. perfringens pathogenesis [2]. Intestinal lesion scores are used to assess the severity of NE [26,27,36] and low scores are indicative of subclinical cases as presented herein. Although CQ15 displayed the lowest recorded lesions in the duodenum during peak infection (d 8), there was not a significant effect on lesions in either the duodenum or jejunum. A decrease in lesions could be an indication of a more intact, healthy, and functioning gut.

Tight junction proteins play an important role in gut integrity and homeostasis and are constantly remodeling in response to external stimuli in the gut lumen such as nutrients and commensal or pathogenic microbes [34,35,36]. The claudin family is a major component of tight junctions because it is an adhesion membrane protein, and several members are receptors for the bacterial toxins produced by C. perfringens [37]. Tight junctions are not the only first line of defense in maintaining the intestinal barrier; MUC2 is produced by goblet cells and is considered a biomarker of intestinal health because it reduces microbial adhesion to the mucosa [38,39]. Coccidia infection has been shown to increase mucus production which can contribute rapid proliferation of C. perfringens by providing protein-rich nutrients because of the damage caused to the host cell [40]. Our findings suggest that supplementing the diet with CQ increased the mRNA abundance of CLDN1 and CLDN5. Claudin 1 and occludin are markers for regulation of the tight junction paracellular permeability barrier, and they help seal the space between two enterocytes avoiding the translocation of any harmful molecule from the intestinal lumen into the underlying tissue and bloodstream [37]. mRNA abundance of JAM3 and MUC2 were lower in the jejunum of supplemented groups opposite to that of ZO2 in the same tissues. This variation in abundance could be associated with better intestinal integrity and lower permeability in the CQ supplemented groups which can also be demonstrated by the reduction in lesions.

Both AMPK and PGC1α are important in regulating energy metabolism within cells and play an essential role in intestinal health [36,41]. While ANXA1, is important in the clearance of inflammation and restoration of mucosal homeostasis [42]. The current findings suggest that dietary supplementation of CQ can increase the abundance of AMPK during peak infection, while slightly lowering the abundance of PGC1α during the recovery period. Since immune responses are energy demanding and tend to divert nutrients from growth resulting in reduced performance [27], a decrease in PGC1α could be the result of nutrients being directed towards growth rather than for mounting an immune response in the CQ supplemented groups. The release of ANXA1 in the epithelial lining during peak infection could prevent the adverse effects caused by NE by inducing mucosal restoration and clearance of inflammation.

An intact epithelium prevents the entry of potential pathogens and results in optimal performance and increases the bird’s ability to better absorb and utilize nutrients [2]. Clarity Q at 30g/MT expressed greater mRNA abundance of EAAT3, GLUT- 2, and PepT-2. Differences in the relative abundance of intestinal transporters such as PepT1, GLUT2, and EAAT3 are known to affect body weight, weight gains, and FCR in broilers [43]. Excitatory amino acid transporter 3 has also shown beneficial in initiating the amino acid dependent cell signaling in the mTOR pathway [40]. Nutrient transporters at the apical membrane of the small intestine are essential in moving nutrients into the enterocytes [44]. During dietary supplementation of saponins, SGLT1 and PepT1 have shown to increase cytoplasmic membrane recruitment, which has a positive effect on body weight and FCR in broilers [45]. In general, PepT1 is predominantly expressed in the small intestine and its expression is upregulated in response to diet, malnourishment, and growth factors and could be a mechanism for using abundant resources or to compensate for the lack thereof [46,47]. This increased activity of disaccharides could also be attributed to an increase substrate presence at the apical membrane and can enhance the development of intestinal villi, which is important for efficient nutrient absorption and digestion [48].

Under this subclinical NE challenge model when broilers were supplemented with Clarity Q, the presented data showed a reduction in duodenal lesion scores on d 8 and a slightly improved FCR during the overall grow-out period. The results also showed a positive modulation in mRNA abundance of several tight junction proteins and nutrient transporter genes. As such, dietary supplementation of Clarity Q can potentially assist birds during an enteric disease challenge.