Evaluation of the Dietary Supplementation of a Formulation Containing Ascorbic Acid and a Solid Dispersion of Curcumin with Boric Acid against Salmonella Enteritidis and Necrotic Enteritis in Broiler Chickens

In the poultry industry, enteric bacterial pathogens pose a threat to intestinal health and can contribute to the transmission of zoonotic diseases [1,2], increased mortality in poultry flocks, reduced feed efficiency, decreased rate of body weight gain and, therefore, increase in total production costs [3,4]. Salmonella infection and necrotic enteritis (NE) produced by Clostridium perfringens (CP) are two significant bacterial diseases in poultry [5,6]. Each year, millions of foodborne salmonellosis cases are reported, resulting in an estimated 155,000 deaths [6]. The most common route of transmission from animals to humans is through contaminated food such as meat, eggs and meat-based products [7,8]. It has also been reported that the presence of salmonellosis has caused significant economic losses in poultry production due to the reduction in overall performance and high mortality in affected flocks [4,9]. Another economically significant disease affecting chicken production is NE induced by CP and occurs in two forms. In its acute clinical form, NE can cause significant flock mortality [10–12] for several days, whereas the subclinical and chronic form can significantly impair performance [13,14]. The economic impact of NE on the worldwide poultry industry was estimated at over five to six billion dollars per year [15]. Thus, controlling enteric bacterial disease in poultry is essential to maintain efficient production and improve food safety [2].

Restrictions on the use of antimicrobials at sub-therapeutic doses in animal production [16] have pressured the poultry industry to look for alternatives to reduce the problems of bacterial resistance and also continue to provide performance benefits, eliminating foodborne pathogens as Salmonella, and reducing the NE incidence. Some of these alternatives include probiotics (yeasts or bacteria), plant derivatives such as essential oils or extracts, organic acids, enzymes and lysozymes [2,17,18].

A recent in vitro study published by our laboratory demonstrated the capability of 1% ascorbic acid (AA) to significantly reduce the concentration of Salmonella Enteritidis (S. Enteritidis) in the compartment that simulates the crop, derived from its acidification capacity, but not in the intestinal compartment since it degrades as the pH increases [19]. Another study showed that broiler chickens supplemented with 0.1% of a solid dispersion of curcumin (CUR) with polyvinylpyrrolidone (PVP) and boric acid (BA, CUR/PVP-BA) resulted in a lower S. Enteritidis recovery in both crop and cecal tonsils (CT) because of a possible synergistic effect between them [20]. Therefore, the purposes of the present study were to evaluate the effect of the prophylactic or therapeutic administration of a 0.1% mixture containing AA and a solid dispersion of CUR with PVP and BA (AA-CUR/PVP-BA) in broiler chickens infected with S. Enteritidis, as well as the impact of the dietary administration of 0.1% AA-CUR/PVP-BA in broilers using a laboratory NE challenge model.

The mixture containing AA and CUR/PVP-BA (AA-CUR/PVP-BA) were prepared in two steps. The first step involved the preparation of the solid dispersion of CUR/PVP-BA (1:1 ratio) as previously described [19]. Subsequently, a mixture of 90% of AA and 10% of microcrystalline cellulose (MCC) pH 10.2 was granulated, dried, sieved and finally associated with the solid dispersion of CUR/PVP-BA. The proportion of each component was 33.3% (1:1:1, AA:CUR/PVP:BA) and the particle size obtained was around 700 μm. The AA-CUR/PVP-BA mixture was mixed into the feed for 15 min using a rotary mixer to obtain the experimental diet with a final concentration of 0.1% (1 g/kg of feed).

The starter feed used in this study was formulated to approximate the nutritional requirements for broiler chickens as recommended by the National Research Council [21], and adjusted to the breeder’s recommendations [22]. No antibiotics, coccidiostats or enzymes were added to the feed (Table 1). All animal handling procedures complied with the Institutional Animal Care and Use Committee (IACUC) at the University of Arkansas, Fayetteville (protocol #15006).

A primary poultry isolate of Salmonella enterica serovar Enteritidis (S. Enteritidis) bacteriophage type 13A, was obtained from the USDA National Veterinary Services Laboratory (Ames, IA, USA). This strain is resistant to 25 μg/mL of novobiocin (NO, catalog no. N-1628, Sigma, St. Louis, MO, USA) and was selected due to its resistance to 20 μg/mL of nalidixic acid (NA, catalog no. N-4382, Sigma, St. Louis, MO, USA) in our laboratory. In the present study, 100 μL of S. Enteritidis from a frozen aliquot were added to 10 mL of tryptic soy broth (TSB, Catalog No. 22092, Sigma, St. Louis, MO, USA) and incubated at 37 °C for 8 h, and passed three times every 8 h to ensure that all bacteria were in log phase as previously described [23]. Post-incubation, bacterial cells were washed three times with sterile 0.9% saline by centrifugation at 1864x g for 10 min, reconstituted in saline, quantified by densitometry with a spectrophotometer (Spectronic 20DC, Spectronic Instruments Thermo Scientific, Rochester, NY, USA) and finally diluted to an approximate concentration of 4 x 10⁴ cfu/mL and 4 x 10⁷ cfu/mL. Concentrations of S. Enteritidis were further verified by serial dilutions and plated on brilliant green agar (BGA, Catalog No. 70134, Sigma, St. Louis, MO, USA) with NO and NA for enumeration of actual cfu used in the experiment.

Two independent trials were conducted to evaluate the prophylactic administration of 0.1% AA-CUR/PVP-BA in reducing the incidence of S. Enteritidis in broiler chickens. In each trial, 30 day-of-hatch male Cobb-Vantress broiler chickens (Fayetteville, AR, USA) were randomly allocated to one of two groups (n = 15 chickens): (1) Group challenged with S. Enteritidis (positive control group, PC) and (2) 0.1% (w/w) AA-CUR/PVP-BA into feed and challenged with S. Enteritidis (AA-CUR/PVP-BA group). Chicks were housed in brooder battery cages, provided with their respective diet and water ad libitum, and maintained at an age-appropriate temperature during the seven days of the experiment. On day six of age, all chicks were orally challenged with 1 x 10⁷ cfu of S. Enteritidis per bird. Chicks were euthanized by CO₂ inhalation 24 h post-S. Enteritidis challenge (Day 7), and samples of crop and CT were taken for S. Enteritidis recovery.

The purpose of experiment 2 was to evaluate the effectiveness of the therapeutic administration of 0.1% AA-CUR/PVP-BA in broiler chickens infected with S. Enteritidis during three and 10 days of treatment. For this, an experiment with 60 one-day-old male Cobb-Vantress broiler chickens (Fayetteville, AR, USA) were challenged with 1 x 10⁴ S. Enteritidis cfu per bird and randomly allocated to one of two groups (n = 30 chickens): (1) Positive control group (PC); (2) 0.1% (w/w) AA-CUR/PVP-BA into the feed (AA-CUR/PVP-BA group). Chicks were housed in brooder battery cages, provided with their respective diet and water ad libitum, and maintained at an age-appropriate temperature during the 10 days of the experiment. On days three and 10 post-S. Enteritidis challenge, 15 chicks from each group were euthanized by CO₂ inhalation, and the crop and CT from 12 birds per group were aseptically collected to evaluate S. Enteritidis recovery. Blood samples were collected from the femoral vein and centrifuged (1000x g for 15 min) to separate the serum for the determination of fluorescein isothiocyanate-dextran (FITC-d) concentration on day 10 post-S. Enteritidis challenge. The concentration of FITC-d administered was calculated based on group body weight at day 9 post-S. Enteritidis challenge. Furthermore, intestinal samples for total intestinal IgA levels were also collected.

In experiment 1 and 2, the crop and CT were homogenized and diluted with saline (1:4 w/v), and 10-fold dilutions were plated on BGA with NO and NA, incubated at 37 °C for 24 h to enumerate total S. Enteritidis colony forming units. Subsequently, the crop and CT samples were enriched in 2 x concentrated tetrathionate enrichment broth and further incubated at 37 °C for 24 h. Enrichment samples were streaked onto Xylose Lysine Tergitol-4 (XLT-4, Catalog No. 223410, BD DifcoTM) selective media for confirmation of Salmonella presence.

This experiment was conducted to evaluate the impact of the dietary administration of 0.1% AA-CUR/PVP-BA on growth performance, intestinal barrier integrity and ileum lesions in broiler chickens using a laboratory necrotic enteritis (NE) challenge model. One hundred and twenty day-of-hatch male Cobb-Vantress broiler chickens (Fayetteville, AR, USA) were randomly assigned to three different groups of four replicates each with ten broiler chickens (n = 40/group): (1) Non-challenged control (negative control group, NC), (2) challenged control (positive control group, PC) and, (3)  challenge control + 0.1% AA-CUR/PVP-BA into the feed (AA-CUR/PVP-BA group). All chicks were raised in floor pens (300 cm x 150 cm) for 21 days, provided with their respective diet and water ad libitum, and maintained at an age-appropriate temperature protocol during the experiment. On day 21, broiler chicks were euthanized by CO₂inhalation, and the right half of the liver from 12 broiler chickens was aseptically collected in sterile sample bags (Nasco, Fort Atkinson, WI, USA) to evaluate bacterial translocation. Additionally, blood samples were collected from the femoral vein and centrifuged (1000 g for 15 min) to separate the serum for FITC-d estimation. The concentration of FITC-d administered was calculated based on group body weight at 20-day-old. Likewise, intestinal samples for the measurement of total intestinal IgA levels were also collected. Ileum NE lesion scores (ILS, n = 25 broiler chickens/group) were evaluated as recommended by Hofacre [24]: 0 = No lesions; 1 = thin-walled and friable intestines; 2 = focal necrosis, gas production and ulceration; 3 = extensive necrosis, hemorrhage and gas-filled intestines; and 4 = generalized necrosis typical of field case, marked hemorrhage. Finally, body weight (BW) and body weight gain (BWG) were evaluated on a weekly basis. Feed intake (FI) and feed conversion ratio (FCR) were obtained at 21-d of age.

NE was induced in the broiler chickens as previously described [25,26] with slight modifications. Briefly, day-old broiler chickens were challenged with a concentration of 1 x 10⁸ cfu of Salmonella Typhimurium (ST) per bird by oral gavage. This organism was isolated from poultry and obtained from the USDA National Veterinary Services Laboratory (Ames, IA, USA). The isolate was resistant to novobiocin (25 μg/mL of NO, catalog no. N-1628, Sigma) and was selected for resistance to nalidixic acid (20 μg/mL of NA, catalog no. N-4382, Sigma) in our laboratory. ST culture was performed in the same way as described above for S. Enteritidis. However, ST suspension was diluted to an approximate concentration of 4 x 10⁸ cfu/mL. The concentration of ST was further verified by serial dilution and plated on brilliant green agar (BGA, Catalog no. 70134, Sigma) with NO and NA for enumeration of actual cfu used in the experiment. Subsequently, at day 13 of age, broiler chickens were challenged with a dose of 2 x 10⁴ sporulated oocysts of Eimeria maxima (EM) per bird by oral gavage. Oocysts were propagated in vivo, according to previously published methods [27,28] and a preliminary dose titration study was carried out, offset by one week, to determine the EM challenge selection for the present study. At day 18 of age, chickens were challenged with a concentration of 1 x 10⁹ cfu of a mixture of two Clostridium perfringens (CP) isolates per bird by oral gavage. Dr. Jack McReynolds (USDA-ARS, College Station, TX, USA) kindly donated the first strain of CP previously described in an NE challenge model [29]. The second strain was isolated from a separate Eimeria challenge experiment in our laboratory with an inadvertent resulting NE (four weeks of age). Then, a single aliquot of each isolate was individually amplified in TSB with thioglycolate (Catalog no. 212081, Becton Dickinson, Sparks, MD, USA) overnight and subsequently mixed. Plating 10-fold dilutions confirmed the concentration of CP on phenylethyl alcohol agar plates (PEA, Becton Dickinson, Sparks, MD, USA) with 5% sheep blood (Remel, Lenexa, KS, USA).

Briefly, liver samples were homogenized, weighed and diluted 1:4 w/v with sterile 0.9% saline enriched with sodium thioglycolate. Then, 10-fold dilutions were plated on tryptic soy agar (TSA, catalog no. 211822, Becton Dickinson, Sparks, MD, USA) with thioglycolate for anaerobic bacteria (AB) recovery. Plates were then incubated anaerobically at 37 °C for 24 h to enumerate entire AB colony forming units per g of tissue.

FITC-d (MW 3–5 kDa; Sigma-Aldrich Co., St. Louis, MO, USA) was used as a marker of paracellular transport and mucosal barrier dysfunction [30,31]. One hour before the chicks were euthanized by CO₂ inhalation, 12 or 20 broiler chickens from each group were given an oral gavage dose of FITC-d (8.32 mg/kg of body weight), and three or five broiler chickens per group were used as controls for the experiment 2 or 3, respectively. The concentrations of FITC-d from diluted sera were measured fluorometrically at an excitation wavelength of 485 nm and an emission wavelength of 528 nm (Synergy HT, Multi-mode microplate reader, BioTek Instruments, Inc., VT, USA) [32].

Total IgA levels in experiments 2 and 3 were determined in 12 gut rinse samples each as previously described [33]. An intestinal section of 5 cm from the Meckel’s diverticulum to the ileocecal junction was taken and rinsed three times with 5 mL of 0.9% saline; then the rinse was collected in a tube and centrifuged at 1864x g at 4 C for 10 min. The supernatant was poured into a 96-microwell plate and stored at −20 °C until tested. A commercial indirect ELISA set was used to quantify IgA according to the manufacturer’s instructions (Catalog No. E30-103, Bethyl Laboratories Inc., Montgomery, TX 77356, USA). 96-well plates (Catalog No. 439454, Nunc MaxiSorp, Thermo Fisher Scientific, Rochester, NY, USA) were used, and samples diluted 1:100 were measured at 450 nm using an ELISA plate reader (Synergy HT, multi-mode microplate reader, BioTek Instruments, Inc., Winooski, VT, USA). Total intestinal IgA levels obtained were multiplied by the dilution factor (100) to determine the amount of chicken IgA in the undiluted samples.

Data from S. Enteritidis and AB counts (Log cfu/g), BW, BWG, FI, FCR, total IgA levels, serum FITC-d concentration and ileum NE lesion score were subjected to an analysis of variance (ANOVA) as a completely randomized design, using the general linear models procedure of Statistical Analysis System (SAS) [34]. The experimental unit for each variable is reported in each table respectively. Significant differences among the means were determined by Duncan’s multiple range test at p < 0.05. Enrichment data were expressed as positive/total chickens (%), and the recovery percentage of AB and S. Enteritidis were compared using the chi-squared test of independence [35], testing all possible combinations to determine the significance (p < 0.05).

The results of the prophylactic administration of 0.1% AA-CUR/PVP-BA on S. Enteritidis colonization in the crop and CT of broiler chickens in trials 1 and 2 (Exp 1) are summarized in Table 2. Although there was no reduction in S. Enteritidis colonization in the crop of chickens treated with 0.1% AA-CUR/PVP-BA into the feed in both trials, in CT, the concentration of S. Enteritidis significantly decreased by more than 1.6 log in comparison with the positive control (PC, p < 0.05). Furthermore, chickens receiving 0.1% of AA-CUR/PVP-BA had a significant reduction in the number of positive S. Enteritidis samples in CT compared to PC.

Table 3 summarizes the effect of the therapeutic administration of 0.1% AA-CUR/PVP-BA in broiler chickens on S. Enteritidis colonization in the crop and CT (experiment 2). On day three post-S. Enteritidis challenge, no significant differences were observed in the concentration of S. Enteritidis in the crop and CT when comparing the PC and treated group. However, a reduction of 2.05 log and 2.71 log in S. Enteritidis concentration was observed in the crop and CT of treated chickens when compared with control group at 10-day post-S. Enteritidis challenge, respectively (Table 3). Furthermore, a significant decrease in serum FITC-d concentration and significantly lower total intestinal IgA levels were observed in broilers treated with 0.1% AA-CUR/PVP-BA when compared to PC on 10-day post-S. Enteritidis challenge (Table 4).

The effect of the dietary inclusion of 0.1% AA-CUR/PVP-BA on growth performance of broiler chickens in the NE model is summarized in Table 5. Seven days post Salmonella Typhimurium (ST) challenge, body weight (BW) and body weight gain (BWG) of the PC and AA-CUR/PVP-BA groups were significantly reduced (≈11 g in both cases) as compared to the negative control (NC) group. However, there were no significant differences in BW and BWG between the NC and AA-CUR/PVP-BA groups in the second week (7–14 d). Although PC was the group with the highest BW and BWG during the second week, the inclusion of 0.1% AA-CUR/PVP-BA into the feed resulted in a numerical increase in BWG (4.7 g) after the Eimeria maxima (EM) challenge (14–18 d) as compared to PC group. After the Clostridium perfringens (CP) challenge (day 18), 0.1% AA-CUR/PVP-BA allowed the chickens to gain 2.88 g from day 18 to day 21, meanwhile PC group reduced its BW in 11.16 g in the same period. During the last week of the trial (14–21 d), a numerical increase in BWG (≈17 g) was observed in the group supplemented with 0.1% AA-CUR/PVP-BA in comparison with the PC group. Interestingly, the feed intake (FI) accumulated (0–21 d) was significantly lower in the AA-CUR/PVP-BA group when compared to the NC and PC groups (Table 5). In the case of the feed conversion ratio (FCR), the PC group (0–21 d) had a significant and numerically lower efficiency ratio compared to the NC and AA-CUR/PVP-BA groups, respectively. Furthermore, the NC group clearly showed significant lower values in ileum lesion scores (ILS), bacterial translocation (BT) and serum FITC-d when compared to the PC or AA-CUR/PVP-BA groups (Table 6). Broilers supplemented with 0.1% AA-CUR/PVP-BA tended to have a reduction in ILS, BT and serum FITC-d concentration, when compared to the PC group (p = 0.07). Interestingly, the PC group showed a significant increase in total intestinal IgA levels when compared to the NC or AA-CUR/PVP-BA groups (Table 6).

Foodborne pathogens and control of avian diseases as NE, remain high priority topics in the poultry industry [36]. However, due to the ban of antibiotic growth promoters by the spread of bacterial resistance to common antibiotics and the incidence of NE, this industry has been actively looking for other equally active molecules to avoid these problems [37,38].

In experiment 1, prophylactic administration of 0.1% AA-CUR/PVP-BA (1:1:1) in broiler chickens was capable to significantly reduce the concentration of S. Enteritidis in CT but not in the crop (Table 2). Previous in vitro and in vivo studies performed in our laboratory showed that 1% AA had the best antimicrobial properties against S. Enteritidis in the compartment that simulates the crop, but not in the intestinal compartment [19], whereas the administration of 0.1% CUR/PVP-BA into the feed significantly decreased the concentration of S. Enteritidis in broiler chickens [20]. Therefore, reduction in S. Enteritidis concentration in CT could only be related to the antimicrobial effect of CUR/PVP-BA derived from the synergistic effect between CUR/PVP and BA.