**Activity and Anti-Aflatoxigenic E**ff**ect of Indigenously Characterized Probiotic Lactobacilli against** *Aspergillus flavus***—A Common Poultry Feed Contaminant**

**Nimra Azeem 1, Muhammad Nawaz 1,\*, Aftab Ahmad Anjum 1, Shagufta Saeed 2, Saba Sana 1, Amina Mustafa <sup>1</sup> and Muhammad Rizwan Yousuf <sup>3</sup>**


Received: 14 March 2019; Accepted: 11 April 2019; Published: 15 April 2019

**Simple Summary:** Mycotoxicosis in poultry has been seriously damaging the poultry production in Pakistan, resulting in economic losses to the country. The present study may act as a preliminary step for exploring the effect of indigenously characterized potential probiotic lactobacilli on aflatoxin production by *Aspergillus flavus*. The present study explored anti-fungal *Lactobacillus* strains. Further investigations revealed their in vitro aflatoxin binding and anti-aflatoxigenic capabilities. These findings demonstrated *L. gallinarum* PL 149 to be an effective binder of aflatoxin B1 which may be used as a biocontrol agent against *A. flavus* and aflatoxin B1 production. It may be further employed for aflatoxin binding in poultry gut after in vivo evaluations.

**Abstract:** Aflatoxin contamination in human food and animal feed is a threat to public safety. Aflatoxin B1 (AFB1) can be especially damaging to poultry production and consequently economic development of Pakistan. The present study assessed the in vitro binding of AFB1 by indigenously characterized probiotic lactobacilli. Six isolates (*Lactobacillus gallinarum* PDP 10, *Lactobacillus reuetri* FYP 38, *Lactobacillus fermentum* PDP 24, *Lactobacillus gallinarum* PL 53, *Lactobacillus paracasei* PL 120, and *Lactobacillus gallinarum* PL 149) were tested for activity against toxigenic *Aspergillus flavus* W-7.1 (AFB1 producer) by well diffusion assay. Only three isolates (PL 53, PL 120, and PL 149) had activity against *A. flavus* W-7.1. The ameliorative effect of these probiotic isolates on AFB1 production was determined by co-culturing fungus with lactobacilli for 12 days, followed by aflatoxin quantification by high-performance liquid chromatography. In vitro AFB1 binding capacities of lactobacilli were determined by their incubation with a standard amount of AFB1 in phosphate buffer saline at 37 ◦C for 2 h. AFB1 binding capacities of isolates ranged from 28–65%. Four isolates (PDP 10, PDP 24, PL 120, and PL 149) also ceased aflatoxin production completely, whereas PL 53 showed 55% reduction in AFB1 production as compared to control. The present study demonstrated *Lactobacillus gallinarum* PL 149 to be an effective candidate AFB1 binding agent against *Aspergillus flavus*. These findings further support the binding ability of lactic acid bacteria for dietary contaminants.

**Keywords:** Aflatoxin B1; *Lactobacillus*; anti-fungal; *Aspergillus flavus*; in vitro; poultry

#### **1. Introduction**

Poultry is one of the major sectors playing a role in the enhanced economic activity of Pakistan but still it faces a lot of problems, including mycotoxicosis. Mycotoxins are toxic secondary metabolites of fungal origin, which can cause various diseases and death in animals and humans. Ergot alkaloids, fumonisins, patulin, aflatoxin, citrinin, trichothecenes, ochratoxin A, and zearalenone are all examples of some different mycotoxins. Aflatoxins, produced by *Aspergillus parasiticus*, *Aspergillus flavus*, and *Aspergillus nomius*, are of great importance because of their biological and biochemical effects on living systems [1]. Aflatoxin-producing molds are globally and can flourish on a variety of food and feed commodities during production, processing, storage, and transportation procedures [1–3]. These molds can infect crops, especially in hot and humid conditions, resulting in economic loss and adverse effects on consumers' health.

Aflatoxin is a potent carcinogen, mutagen, contains hepatotoxic and immunosuppressant effects and inhibit several metabolic systems resulting in liver and kidney damage [1,4]. Aflatoxin and citrinin cause increased fragility of the vascular system and produce hemorrhages in body tissues. Among aflatoxins, aflatoxin B1 is the most potent, and it is categorized among class 1 human carcinogens. Different factors including pH, temperature, water activity, available nutrients, and competitive inhibition by other microorganisms can affect aflatoxin production in feed [3]. Appropriate harvesting and storage conditions of crops and feed play important roles in aflatoxin reduction.

Various methods have been employed for the removal or inactivation of aflatoxins, including physical, biological, and chemical methods. Chemical treatments may include roasting, ammoniation, and other solvent extraction techniques. Many aflatoxin binders, like activated carbon and various mineral clays, are commercially available and act as sequestering agents and tightly bind aflatoxin; the resulting binding complex is then excreted from the animal's body [5]. These toxin binders can restore the nutritional value of the feed, but these chemical methods are unsafe, unhealthy, and expensive [6]. Toxin removal by microorganisms is a promising and economical method for decontaminating raw materials and food [7]. Numerous investigations have reported the inhibitory effects of microbes including actinomycetes, yeast, mold, and bacteria on mold growth and aflatoxin production [3]. Thus, beneficial microorganisms may serve as an alternative therapy for mycotoxicosis.

Anti-mutagenic lactic acid bacteria can remove mutagens from food by physical means [8]. Toxin binding by bacteria occurs through cell wall components, namely polysaccharides or polypeptides. Many researchers have studied this binding mechanism, but the exact mechanism of binding is still unknown [9].

Researchers are paying more attention towards preventing the absorption of aflatoxins in the gastrointestinal tracts of users by the aid of probiotic bacterial supplements in food and feed [10]. According to the World Health Organization (WHO), probiotics are defined as live microorganisms which when administered in adequate amounts exert healthy effects to host [11]. *Lactobacillus*, *Bifidobacterium*, *Enterococcus*, *Saccharomyces*, and *Bacillus* may serve as probiotics.

Lactobacilli can efficiently remove aflatoxins from contaminated broth. The toxin removal mechanism involves sequestration by binding the toxin to the cell wall instead of metabolic degradation [12]. The present study may act as a preliminary step for studying the effect of indigenously characterized potential probiotic lactobacilli on aflatoxin production by *Aspergillus flavus*, so that lactobacilli can be used as biocontrol agents. The present study also assessed the in vitro AFB1 binding capacity of *Lactobacillus* spp., so that these probiotic strains can be employed as toxin binders in place of chemicals in animal feed and thereby the harmful effects of chemical toxin binders can be avoided.

#### **2. Materials and Methods**

#### *2.1. Identification of Isolates*

Previously characterized probiotic lactobacilli (*n* = 6) of poultry and fermented food origin [13] and toxigenic *Aspergillus flavus* W-7.1 were procured from the Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, as listed in Table 1. Lactobacilli were revived using De Man, Rogosa, and Sharpe (MRS) agar and identified as describe previously [14]. Fungal strain was cultured on Sabouraud Dextrose Agar (SDA) medium incubated at 37 ◦C for 5–6 days. Culture and microscopic characters were observed for identification as described previously [15].


**Table 1.** Antifungal activity of cell free supernatants of lactobacilli.

NZ: No zone of inhibition.

#### *2.2. Antifungal Activity of Lactobacilli*

Antifungal activity of lactobacilli (*n* = 6) was determined by well diffusion assay as described elsewhere [16]. Briefly, SDA medium seeded with fungal spores (10<sup>7</sup> spores/mL) was poured into sterile Petri dishes and allowed to solidify. Wells were punctured in the medium which were then sealed with sterile molten agar. Cell free supernatant (100 μL) of each lactobacilli strain was added into the respective wells. After 3–4 days incubation at 28 ◦C aerobically, the diameter of zones of inhibition (mm) was measured.

#### *2.3. E*ff*ect of Lactobacilli on Aflatoxin Production*

The effect of lactobacilli on aflatoxin production by *Aspergillus flavus* was observed by inoculating 1 mL bacterial suspension (1 McFarland) in yeast extract sucrose broth (YESB) supplemented with a standard amount of fungal spores (107 spores/mL), followed by incubation at 28 ◦C and 100 rpm for 10 days. YESB media supplemented with known fungal spores and plain YESB media without any inoculation were also incubated as positive and negative controls, respectively. After incubation, medium containing lactobacilli and fungus was filtered through Whatman filter paper no 1 and aflatoxin B1 quantity in filtrate was measured by high-performance liquid chromatography (HPLC) and compared with controls [6]. Aflatoxin B1 was detected by HPLC and quantified using the following formulae:

$$\text{Quantity of Aflatoxins} \left(\frac{\text{ng}}{\text{mL}}\right) = \frac{\text{peak area of sample}}{\text{peak area of standard}} \times 100\tag{1}$$

$$\% \text{ age reduction} = \frac{1 - \text{(Peak area of AFB1 in treatment)}}{\text{(Peak area of AFB1 in control)}} \tag{2}$$

#### *2.4. Aflatoxin B1 Extraction*

For toxin extraction, a previously established protocol was used with modifications [17]. Briefly, broth culture of *Aspergillus flavus* was autoclaved at 121 ◦C and 15 psi and then homogenized using homogenizer. Twenty-five grams of homogenate was treated with chloroform (90 mL), methanol (10 mL), NaCl (5 g), and distilled water (10 mL) and incubated at 37 ◦C with continuous shaking

(150–160 rpm) for 30 min. Filtration was carried out using Whatman filter paper #4 and filtrate was concentrated in a water bath at 50 ◦C. Concentrate was ground to fine powder and reconstituted in 3 mL chloroform volume and stored at 4 ◦C.

#### *2.5. Toxin Binding Assay*

Standard aflatoxin B1 solution was prepared by the method described elsewhere [18]. Prepared standard aflatoxin solution was then added to sterile phosphate buffer saline (PBS) containing lactobacilli culture (1 McFarland). After 2 h of incubation, cells with bound toxin were separated by centrifugation at 10,000 rpm for 5 min and unbound aflatoxin in supernatant was quantified by HPLC.

#### *2.6. High Performance Liquid Chromatography (HPLC)*

Aflatoxins were quantified by Agilent HPLC system, 1100 series (Agilent, Santa Clara, CA, USA) as described previously [19]. A mixture of acetonitrile, water, and methanol was used as mobile phase at a flow rate of 1 mL per minute. Mobile phase was firstly purified using a filtration assembly and then sonicated for 10 min at 20 ◦C in order to avoid gas bubbles. Next, 20 μL samples were injected using a micro-syringe. After 15 min, ultra violet (UV) absorbance was recorded at 254 nm. Sample peaks were analyzed and compared with standard UV absorption data of secondary metabolites at various retention times. Limit of detection (LOD) and limit of quantification (LOQ) of standard aflatoxin were 0.01 ng/mL–100 μg/mL and 0.1 ng/mL–100 μg/mL, respectively.

#### *2.7. Statistical Analysis*

Mitigation of aflatoxin production and toxin binding capacity of lactobacilli was compared by one-way ANOVA (analysis of variance) followed by Turkey's multiple comparison test using Graph pad prism 5.0 software (GraphPad Software, San Diego, CA, USA).

#### **3. Results**

A total of six potential probiotic lactobacilli, including *Lactobacillus gallinarum* PDP 10, *Lactobacillus reuteri* PDP 24, *Lactobacillus fermentum* FYP 38, *Lactobacillus gallinarum* PL 53, *Lactobacillus paracasei* PL 120, and *Lactobacillus gallinarum* PL 149, were procured from the Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan. All isolates were Gram-positive rods and catalase negative.

Only three isolates (PL 53, PL 120, and PL 149) had antifungal activity observed by well diffusion assay, as illustrated in Table 1 and Figure 1.

**Figure 1.** Activity of cell free supernatant of *Lactobacillus gallinarum* PL 149 against *Aspergillus flavus.*

Four isolates (PDP 10, PDP 24, PL 120, and PL 149) showed 100% removal of AFB1, PL 53 caused 55.2% reduction, while FYP 38 showed an enhancing effect on aflatoxin B1 production, as described in Table 2. All isolates showed a varied degree of toxin binding capacities, as described in Table 3 and Figure 2. PL 149 was the most effective binder of aflatoxin B1, with 65% capacity.


**Table 2.** Effect of lactobacilli on aflatoxin B1 production.

AFB1: Aflatoxin B1; ND: Not detected.

**Table 3.** Aflatoxin B1 binding capacity of probiotic lactobacilli.

**Figure 2.** High-performance liquid chromatography chromatograms of aflatoxin B1 present in control and suspension after treatment with lactobacilli: (**a**) Control; (**b**) PDP 10; (**c**) FYP 38; (**d**) PL 149.

#### **4. Discussion**

Aflatoxins represent a group of fungal secondary metabolites that are of great health and economic importance. In developing countries, greater than five billion people are at risk of chronic exposure to aflatoxins, which are capable of causing liver cancer [4]. Consequently, there is an increasing demand for novel preventive and controlling strategies for aflatoxin contaminations in food and feed. Recent studies have revealed the aflatoxin binding ability of lactobacilli. Many bacteria have been reported as aflatoxin binders, including *Flavobacterium aurantiacum*, *L. plantarum*, *L. pentosus*, and *L. beveris* [20–22]. Likewise, *Lactobacillus casei psuedoplantarum* 371, obtained from silage inoculum, inhibited aflatoxin B1 and G1 synthesis by *Aspergillus flavus* subsp*. parasiticus* NRRL 2999 in liquid medium [23]. In a previous study, a mixture of lactobacilli was found to reduce mold growth, germination of spores, and production of aflatoxins by *Aspergillus flavus* subsp. *parasiticus* [24]. A large number of such studies have been reported worldwide, but few related studies have been reported in Pakistan.

The present study can act as a preliminary step in a multistep study to investigate the anti-fungal, anti-aflatoxigenic, and in vitro AFB1 binding capacities of previously characterized indigenous phytase-solubilizing probiotic lactobacilli spp. of poultry and fermented food origin [13] against toxigenic *Aspergillus flavus*. This study identified three probiotic lactobacilli isolates (*Lactobacillus gallinarum* PL 53*, Lactobacillus paracasei* PL 120*,* and *Lactobacillus gallinarum* PL 149) as antifungal agents. Such inhibitory effects may be a result of lactic acid production or physical interaction of lactobacilli with mold. Similar inhibitory effects of *L. acidophilus* ATCC 4495 and *L. brevis* were also demonstrated previously against *Aspergillus flavus* and *Aspergillus parasiticus*, respectively [25,26].

Four isolates (PDP 10, PDP 24, PL 120, PL 149) in the present study ceased aflatoxin production completely, whereas PL 53 showed 55% reduction. On the other hand, FYP 38 showed an enhancing effect on aflatoxin B1 production. These variable results may depict different bacterial cell wall structures. Many other investigators have reported similar results, in which various lactic acid-producing bacteria, including *Lactobacillus*, were capable of inhibiting aflatoxin production, whereas some lactic acid bacteria, like *Lactococcus lactis*, stimulated aflatoxin biosynthesis [27]. Cell wall polysaccharides and peptidoglycans have been considered bacterial tools for mycotoxin binding [28]. Extracellular metabolites of *Lactobacillus casei* KC 324 has been reported to mitigate mold growth and aflatoxin production of *Aspergillus flavus* ATCC 15517 [29]. Commercial silage was once reported to contain inhibitory lactobacilli against aflatoxin B1 and G1 production [30]. *L. plantarum* ATCC 4008*, L. plantarum* 12006, *Lactobacillus plantarum* 299V, *L. paracasei* subsp. *paracasei* LMG 13552, and *L. rhamnosus* VT1 reduced aflatoxin production by 85–92% to 96.3–98.3% [31], whereas in the present study a 100% reduction in AFB1 production by *L. gallinarum* PDP 10 and PL 149, *L. reuteri* PDP 24, and *L. paracasei* PL 120 was observed. It may also be a result of very low aflatoxin production in control conditions as well. Yeast can also act as an effective biocontrol agent against aflatoxins. *S. boulardii* and *S. cerevisiae* individually reduced aflatoxin production by 72.8% and 65.8%, respectively, while their combinations reduced aflatoxin production from 71.1% to 96.1%. Supplementation of peanut grains with combinations of *S. boulardii* plus *L. delbrueckii*, *S. boulardii* and *S. cerevisiae*, *L. delbrueckii* and *S. cerevisiae* showed reduction by 96.1%, 66.7%, and 71.1%, respectively [32]. *Lactobacillus fermentum* PTCC 1744 and *Bifidobacterium bifidum* PTCC 1644 were also reported to reduce aflatoxin production by more than 99% in comparison with controls [6], although this report is contradictory to the present research which revealed the enhancing effect of *Lactobacillus fermentum* on AFB1 production by *A. flavus*.

In the present study, *Lactobacillus gallinarum* PDP 10, *Lactobacillus fermentum* FYP 38, Lactobacillus reuteri PDP 24, *Lactobacillus gallinarum* PL 53, *Lactobacillus paracasei* PL 120, and *Lactobacillus gallinarum* PL 149 showed aflatoxin binding capacities of 51.3%, 56%, 2%, 42%, 28%, and 65%, respectively. These results were quite similar with that of Fazeli et al. [33]. In a previous study, the aflatoxin B1 binding capacities of *Lactobacillus* and *Bifidobacterium* strains were assessed, which were found to range from 5.8% to 31.3% [12]. On the other hand, the present study reported up to 65% AFB1 binding abilities of probiotic lactobacilli. A previous study reported that *Lactobacillus casei* had a 20% AFB1 binding capacity [34], which is less than that of *L. paracasei* PL 120 (28%), whereas *Lactobacillus delbrueckii* subsp. *lactis* was reported to have the maximum antifungal (67.43% reduction) and anti-aflatoxigenic (94.33% reduction) activity against *A. flavus* [35]. Another previous study reported 43.9–64.2% aflatoxin degrading ability of lactobacilli strains [36]. Past investigations revealed similar responses of non-viable and viable cells of *Enterococcus faecium* strains, whose binding abilities were insignificant statistically. Hence, it was hypothesized that AFB1 detoxification of *Enterococcus faecium* is a result of aflatoxin binding to bacterial cell wall; a similar mechanism has been also described by other relevant studies [37]. An in vivo experiment revealed the neutralizing capability of *Lactobacillus casei Shirota* on AFB1 toxicity on the intestine and body weight of host via binding processes [38]. Thus, lactic acid bacteria have been declared as good candidates to prevent aflatoxicosis in farm animals and poultry [9].

#### **5. Conclusions**

The present study reported the anti-fungal, anti-aflatoxigenic, and AFB1 binding capacity of six indigenously characterized probiotic strains. It was concluded that *L. gallinarum* PL 149 may inhibit the AFB1 production by *A. flavus* and also bind AFB1. *L. gallinarum* PL 149 may be employed for aflatoxin binding in poultry gut after in vivo evaluations.

**Author Contributions:** Conceptualization: M.N. and A.A.A.; methodology: M.N., A.A.A., N.A., and S.S. (Shagufta Saeed); software: M.N.; validation: M.N., A.A.A., M.R.Y., S.S. (Shagufta Saeed), and S.S. (Saba Sana); formal analysis: M.N., N.A., M.R.Y., A.M., and S.S. (Saba Sana); investigation: N.A., A.M., and S.S. (Shagufta Saeed); resources: M.N. and A.A.A.; data curation: N.A. and A.M.; writing—original draft preparation: A.M. and N.A.; writing—review and editing: A.M., N.A., S.S. (Saba Sana), M.R.Y., and M.N.; visualization: M.N., A.A.A., S.S. (Shagufta Saeed), and N.A.; supervision: M.N., A.A.A., S.S. (Shagufta Saeed), and S.S. (Saba Sana); project administration: M.N. and A.A.A.; funding acquisition: M.N. and A.A.A.

**Funding:** This project was partially supported through Higher Education Commission (HEC) project No. 4333/NRPU/R&D/HEC/14/278 and NRPU Project # 4148.

**Conflicts of Interest:** The authors declare no conflict of interest. The funding agency had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **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**

**Daniel Hernandez-Patlan 1, Bruno Solís-Cruz 1, Karine Patrin Pontin 2, Juan D. Latorre 3, Mikayla F. A. Baxter 3, Xochitl Hernandez-Velasco 4, Ruben Merino-Guzman 4, Abraham Méndez-Albores 5, Billy M. Hargis 3, Raquel Lopez-Arellano <sup>1</sup> and Guillermo Tellez-Isaias 3,\***


Received: 3 April 2019; Accepted: 18 April 2019; Published: 22 April 2019

**Simple Summary:** Prophylactic or therapeutic administration of a 0.1% mixture containing ascorbic acid (AA) and a solid dispersion of curcumin (CUR) with polyvinylpyrrolidone (PVP) and boric acid (BA) (AA-CUR/PVP-BA) significantly reduced the concentration of *Salmonella* Enteritidis in broiler chickens and had a positive effect in slightly diminishing the negative impact of necrotic enteritis (NE).

**Abstract:** Two experiments were conducted to evaluate the effect of the prophylactic or therapeutic administration of a 0.1% mixture containing ascorbic acid and a solid dispersion of curcumin with polyvinylpyrrolidone and boric acid (AA-CUR/PVP-BA) against *Salmonella* Enteritidis (*S*. Enteritidis) in broiler chickens. A third experiment was conducted to evaluate the impact of the dietary administration of 0.1% AA-CUR/PVP-BA in a necrotic enteritis (NE) model in broiler chickens. The prophylactic administration of 0.1% AA-CUR/PVP-BA significantly decreased *S*. Enteritidis colonization in cecal tonsils (CT) when compared to the positive control group (PC, *p* < 0.05). The therapeutic administration of 0.1% AA-CUR/PVP-BA significantly reduced the concentration of *S*. Enteritidis by 2.05 and 2.71 log in crop and CT, respectively, when compared with the PC on day 10 post-*S*. Enteritidis challenge. Furthermore, the serum FITC-d concentration and total intestinal IgA levels were also significantly lower in chickens that received 0.1% AA-CUR/PVP-BA. Contrary, the PC group showed significantly higher total intestinal IgA levels compared to the negative control or AA-CUR/PVP-BA groups in the NE model. However, 0.1% AA-CUR/PVP-BA showed a better effect in reducing the concentration of *S*. Enteritidis when compared to the NE model. Further studies

with higher concentration of AA-CUR/PVP-BA into the feed to extend these preliminary results are currently being evaluated.

**Keywords:** chickens; ascorbic acid; curcumin; boric acid; necrotic enteritis; *Salmonella* Enteritidis

#### **1. Introduction**

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.

#### **2. Materials and Methods**

#### *2.1. Preparation of Treatments and Diets*

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).

**Table 1.** Ingredient composition and nutrient content of a basal starter diet used in the experiments on as-is basis.


<sup>1</sup> Poultry fat West Coast Reduction LTD is primarily obtained from the tissue of poultry in the commercial process of rendering or extracting. This finished product was used as an energy source for animal and aquaculture feed. <sup>2</sup> Vitamin premix supplied per kg of diet: Retinol, 6 mg; cholecalciferol, 150 μg; dl-α-tocopherol, 67.5 mg; menadione, 9 mg; thiamine, 3 mg; riboflavin, 12 mg; pantothenic acid, 18 mg; niacin, 60 mg; pyridoxine, 5 mg; folic acid, 2 mg; biotin, 0.3 mg; cyanocobalamin, 0.4 mg. <sup>3</sup> Mineral premix supplied per kg of diet: Mn, 120 mg; Zn, 100 mg; Fe, 120 mg; copper, 10 mg to 15 mg; iodine, 0.7 mg; selenium, 0.2 mg; and cobalt, 0.2 mg. <sup>4</sup> Ethoxyquin.

#### *2.2. Salmonella Strain and Culture Conditions*

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 1864× *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 <sup>×</sup> 104 cfu/mL and 4 <sup>×</sup> 10<sup>7</sup> 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.

#### *2.3. Experiment 1*

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 <sup>×</sup> 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.

#### *2.4. Experiment 2*

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 <sup>×</sup> <sup>10</sup><sup>4</sup> *<sup>S</sup>*. 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 (1000× *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.

#### *2.5. Salmonella Recovery*

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 × 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.

#### *2.6. Experiment 3*

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 × 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 CO2 inhalation, 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.

#### *2.7. NE Model: Challenge or Ganisms*

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 <sup>×</sup> 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 <sup>×</sup> 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 <sup>×</sup> 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 <sup>×</sup> 10<sup>9</sup> 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).

#### *2.8. Liver Bacterial Translocation (BT)*

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.

#### *2.9. Serum Determination of FITC-d Leakage*

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].

#### *2.10. Total Intestinal Immunoglobulin A (Iga) Levels*

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 1864× *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.

#### *2.11. Data and Statistical Analysis*

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).

#### **3. Results**

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 2.** Prophylactic administration of a 0.1% mixture containing ascorbic acid (AA) and a solid dispersion of curcumin with polyvinylpyrrolidone (CUR/PVP, ratio 1:9) and boric acid (BA) (AA-CUR/PVP-BA) on crop and cecal tonsils (CT) colonization of *Salmonella* Enteritidis (*S*. Enteritidis) <sup>1</sup> in broiler chickens (Experiment 1).


Data are presented in Log cfu/g of tissue. Mean ± SE from 12 chickens. <sup>1</sup> Chickens were orally gavaged with 107 cfu of *S*. Enteritidis per chicken at six-day old, samples were collected 24 h later. a,b Values within treatment columns for each treatment with different superscripts differ significantly (*p* < 0.05). For *S*. Enteritidis incidence, data are presented as positive/total chickens (percentage). \* *p* < 0.05; \*\* *p* < 0.01.

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).

**Table 3.** Therapeutic administration of a 0.1% mixture containing ascorbic acid (AA) and a solid dispersion of curcumin with polyvinylpyrrolidone (CUR/PVP, ratio 1:9) and boric acid (BA) (AA-CUR/PVP-BA) on crop and cecal tonsils (CT) colonization of *Salmonella* Enteritidis (*S*. Enteritidis) <sup>1</sup> in broiler chickens at three and ten days post-*S*. Enteritidis challenge (experiment 2).


Data are presented in Log cfu/g of tissue. Mean ± SE from 12 chickens. <sup>1</sup> Chickens were orally gavaged with 104 cfu of *<sup>S</sup>*. Enteritidis per chicken at 1-day old, samples were collected three and ten days post-*S*. Enteritidis challenge. a,b Values within treatment columns for each treatment with different superscripts differ significantly (*<sup>p</sup>* <sup>&</sup>lt; 0.05). For *S*. Enteritidis incidence, data are presented as positive/total chickens (percentage). \* *p* < 0.05.

**Table 4.** Therapeutic administration of a 0.1% mixture containing ascorbic acid (AA) and a solid dispersion of curcumin with polyvinylpyrrolidone (CUR/PVP, ratio 1:9) and boric acid (BA) (AA-CUR/PVP-BA), on serum concentration of fluorescein isothiocyanate-dextran (FITC-d), and total intestinal immunoglobulin A (IgA) levels in broiler chickens on day ten post *Salmonella* Enteritidis (*S*. Enteritidis) challenge <sup>1</sup> (experiment 2).


Data expressed as mean ± SE from 12 chickens. <sup>1</sup> Chickens were orally gavaged with 104 cfu of *S*. Enteritidis per chicken at 1-d old, samples were collected ten days post-*S*. Enteritidischallenge. a,b Values within columns with different superscripts differ significantly (*p* < 0.05).

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).

**Table 5.** Evaluation of body weight (BW), body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) in broiler chickens consuming a diet supplemented with or without a 0.1% mixture containing ascorbic acid (AA) and a solid dispersion of curcumin with polyvinylpyrrolidone (CUR/PVP, ratio 1:9) and boric acid (BA) (AA-CUR/PVP-BA) on a Necrotic enteritis challenge model <sup>1</sup> (Experiment 3).


<sup>1</sup> Data expressed as mean ± SE from 40 chickens. a,b Values within columns with different superscripts differ significantly (*p* < 0.05).

**Table 6.** Evaluation of a 0.1% mixture containing ascorbic acid (AA) and a solid dispersion of curcumin with polyvinylpyrrolidone (CUR/PVP, ratio 1:9) and boric acid (BA) (AA-CUR/PVP-BA) on ileum NE lesion scores (ILS), bacterial translocation (BT) to the liver, serum concentration of fluorescein isothiocyanate–dextran (FITC-d) and immunoglobulin A (IgA) levels in broiler chickens 1.


<sup>1</sup> Data expressed as mean ± SE. <sup>2</sup> ILS was evaluated in 25 broiler chickens. <sup>3</sup> BT was expressed in Log10 cfu /g of tissue from 12 chickens. <sup>4</sup> FITC-d concentration of 20 serum samples. <sup>5</sup> IgA levels determined in 12 intestine samples. a,b Values within treatment columns for each treatment with different superscripts differ significantly (*p* < 0.05).

#### **4. Discussions**

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.

In the second experiment, the concentration of *S*. Enteritidis in the crop and CT of broiler chickens treated with 0.1% AA-CUR/PVP-BA for 10 days post-*S*. Enteritidis challenge was significantly reduced. Probably, the decrease of *S*. Enteritidis in the crop was due to the combination of the acidifying effect of AA (0.033% in the mixture) given the release of protons in the medium (pKa = 4.1 and 11.6) [8] and the antimicrobial effect of CUR/PVP-BA (0.066% in the mixture). However, the decrease of *S*. Enteritidis in CT was closely related to the antimicrobial effect of CUR/PVP-BA since it has been reported that AA is not capable of acidifying the intestine of chicks, even when administered at 1% into the feed [39] and it is also unstable at a neutral pH [40]. The effectiveness of CUR/PVP-BA in *S*. Enteritidis reduction could be due to the improvement in the solubility and stability of CUR for its association with PVP compared to CUR alone and its interaction with BA to form complexes with better antimicrobial properties [20,41], as well as to the higher residence time in the intestine.

Gut integrity is essential to maintain health and performance of animals [42]. *Salmonella* infections are associated with inflammation and alterations in gut permeability [43–45]. The results in Table 4 show that chickens treated with 0.1% AA-CUR/PVP-BA had both lower serum FITC-d concentration and total intestinal IgA levels compared to PC on day 10 post-*S*. Enteritidis challenge. FITC-d is a marker for evaluating intestinal permeability since it is a high molecular weight molecule (3–5 kDa) that is not permeable under standard conditions [32,46]. The increase in the secretion of IgA provides a critical mucosal immunity [47]. Therefore, these results confirm the decrease in the severity of *S*. Enteritidis infection given the antimicrobial activity of 0.1% AA-CUR/PVP-BA. Total intestinal IgA levels were not evaluated in experiment 1 since it has been reported that in early phases of *S*. Enteritidis infection, there are no significant differences in the secretion of intestinal IgA between infected and uninfected chickens [48,49].

Considering that the prophylactic or therapeutic administration of 0.1% AA-CUR/PVP-BA significantly reduced the concentration of *S*. Enteritidis in CT of broilers, in the third experiment the impact of the dietary administration of 0.1% AA-CUR/PVP-BA using a NE model in broiler chickens was evaluated. Many predisposing factors in NE, such as the ST and EM infection, increased the colonization and proliferation of CP, and the subsequent release of protein toxins that cause intestinal damage [25,50–52]. During the first seven days after the ST challenge, BW of PC and AA-CUR/PVP-BA groups was significantly reduced (*p* < 0.05) as compared to NC (Table 4), confirming that ST had a negative impact in BW [25]. However, in the second week, there were no significant differences in BW and BWG when comparing NC and AA-CUR/PVP-BA groups. Probably, the concentration of ST decreased due to the acidifying effect of AA and the antimicrobial effect of CUR/PVP-BA as described in experiment 2.

Although the *Eimeria* and CP challenges had adverse effects on performance parameters in PC group and in broiler chickens treated with 0.1% AA-CUR/PVP-BA in comparison with NC group, dietary administration of 0.1% AA-CUR/PVP-BA numerically improved FCR (0–21 d) when compared to PC group. Furthermore, broilers supplemented with 0.1% AA-CUR/PVP-BA did not show a reduction in BW after CP challenge as the PC group did, and tended to have a reduction on ILS, BT and serum FITC-d concentration, when compared to the PC group (*p* = 0.07). It has been reported that CUR has anticoccidial properties [53–56]. However, the anticoccidial mechanism of CUR has remained, but it has been proposed that it involves the induction of oxidative stress in coccidia, as well as neutralization of reactive oxygen species [2,57]. Despite that CUR/PVP was associated with BA (CUR/PVP-BA), the anticoccidial effect of CUR is not lowered since the boron molecules interact with the keto-enol groups of CUR [37,58,59] without affecting its phenolic groups, which are responsible for its anticoccidial properties [60]. Therefore, the results obtained in this experiment suggest that

0.1% AA-CUR/PVP-BA into the feed had a positive effect in slightly diminishing the effects of coccidia, a well-documented predisposing factor in NE.

Interestingly, chickens of the PC group showed a significant increase in total intestinal IgA levels when compared to NC and AA-CUR/PVP-BA groups (Table 4). Secretory IgA (SIgA) is an essential part of the adaptive humoral immune system and the primary immunoglobulin that neutralizes pathogens on external mucosal surfaces [33,61,62]. Hence, the significant decrease of IgA levels in the group supplemented with 0.1% AA-CUR/PVP-BA could be related to the anti-inflammatory properties of CUR and BA. While CUR reduces the inflammatory responses by regulating the production of some proinflammatory cytokines [56,63], BA has the ability to reduce levels of inflammatory biomarkers as TNF-α and IL-6 [64,65].

#### **5. Conclusions**

In conclusion, prophylactic or therapeutic administration of 0.1% AA-CUR/PVP-BA significantly reduced the concentration of *S*. Enteritidis in broiler chickens. Furthermore, dietary administration of 0.1% AA-CUR/PVP-BA had a positive effect in slightly diminishing the negative impact of NE. However, 0.1% AA-CUR/PVP-BA showed a better effect in reducing the concentration of *S*. Enteritidis when compared to the NE model. Further studies with a higher concentration of AA-CUR/PVP-BA into the feed to extend these preliminary results are currently being evaluated.

**Author Contributions:** Conceptualization, G.T.-I., A.M.-A., D.H.-P. and B.S.-C.; Formal analysis, D.H.-P., R.M.-G., A.M.-A., B.M.H. and G.T.-I.; Methodology Investigation K.P.P., J.D.L. and M.F.A.B.; Supervision, D.H.-P., B.S.-C. and G.T.-I.; Writing—original draft, D.H.-P., X.H.-V., R.M.-G. and A.M.-A.; Writing—Review & editing, X.H.-V., B.M.H., R.L.-A. and G.T.-I.

**Funding:** This research was supported by the Arkansas Bioscience Institute under the project: development of an avian model for evaluation early enteric microbial colonization on the gastrointestinal tract and immune function.

**Acknowledgments:** The authors thank the CONACyT for the doctoral scholarship number 270728 and the financial support obtained through the program PAPIIT IN218115 of DGAPA-UNAM.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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