Evaluation of the role of green synthesis silver nanoparticles as adsorbents and protective agents for broilers tissue treated with aflatoxin

Abstract


Introduction
Aspergillus. flavus and A. parasiticus are the significant fungal microorganisms that produce aflatoxin, which are secondary toxic metabolites that contaminate poultry feed under high humidity and temperature (1)(2)(3). Aflatoxin B1 (AFB1) is the most hazardous of the critical aflatoxins (4). Additionally, it is regarded as a Group I carcinogen. Because the administration of AFB1 has adverse hepatotoxic, mutagenic, carcinogenic, and teratogenic effects on people and many species of livestock, even fish (5), major economic losses are posed by human and animal consumption of feed or food products affected by this toxin (6), the permissible dose for poultry is 20 ppb. The critical economic impact of aflatoxicosis in broilers is the reduction in body weight gain and growth performance, which is likely brought on by changes in protein metabolism (7)(8)(9). Nanotechnology is promising and innovative, with a wide range of applications, and has great potential benefits in the poultry industry (10). Nanoparticles have unique physical and chemical properties, such as their small size ranging from 1-100 nm and large specific surface area SSA, they are more bioavailability, stable, and bioactivity than bulk particles (11), and therefore they are widely used in drug delivery, vaccine preparation, immune stimulation, encapsulated bioactive compound as well as their activity as antifungal, anti-parasitic, antiviral and antibacterial in addition anti mycotoxin (12)(13)(14)(15). There are physical and chemical methods for manufacturing nanoparticles, and recently, biological methods based on microorganisms (fungal and bacteria) and plant extracts have been used. This method is called green synthesis. It is environmentally friendly because it produces nanomaterials that are pure and less toxic compared to chemical methods and less economical (16). Abdel Ghany (17) refers to the robust activity of silver nanoparticles mediated by cinnamon oil as an antifungal and a decline in toxin production.
So, this study aimed to evaluate the silver nanoparticle (Ag NP) activity mediated by peel pomegranate as improving growth performance, biochemical, and repair tissue injury in the broiler with aflatoxicosis.

Ethical prove
Scientific Ethical Committee on Animal Experimentation at College of Veterinary Medicine, University of Mosul, UM.VET.2022.015.

House and animal management
In this investigation, 45 unsexed broiler Ross 308 1-dayold get from a local hatchery were used and transported to a cage 2x1.5 m 2 in the form of the house care animal in College of Veterinary Medicine-University of Mosul. This farm was declared safe and disinfected with formaldehyde gas (produced by combining 40% formalin with potassium permanganate powder) and kept in a well-ventilated chamber and electric bulbs 200 watts were used in each partition to keep the room at the light and dark cycle for 23/1 hrs./day. Fresh, clean straw had been placed over each cell to create a deep litter 4 centimeters deep. Moreover, there was an appropriate feeder and waterer in each compartment. The chicks were weight as soon as they arrived farm. Newcastle disease and Evan Influenza were given to birds as vaccines (Intervert -injected S.C.), and water and food were available during the experimental 21 days. Preparation of silver nanoparticles according to Al-Othman et al. (18)

Pomegranate peels aqueous extract PPE
Pomegranate peel was obtained from a local supermarket, washed with deionized distilled water, and the drying at 50 °C for 72 hours, then with a blender, crushed into powder, 100 ml of sterile, deionized water and 10 g of peel powder were combined, heated for 15 minutes, and then filtered using Whatman filter paper No. 1.

Synthesis of silver nitrate nanoparticles mediated by PPE
100 ml of silver nitrate 1 mM solution was combined with 10 ml of PPE before being incubated at 60-70 o C for 15-20 minutes. The characterization result of AgNPs prepared as in table 1 (19).

Measurements techniques Characterization of NPs Color
Dark-Brown after three days UV-visible Spectroscopy The peak at the wavelength of 450 nanometers FTIR 451.34 cm -1 is the band for silver nanoparticles binding with O 2 from hydroxyl groups SEM The average size was 40nm Zeta potential The stability of AgNPs at degree -29 mV Prepare a diet contaminated with AFB1 AFB1 was obtained from inoculated Aspergillus flavus in the rice medium and incubated at 28°C for at least two weeks. Following a successful fermentation, the rice was steam heated to inhibit fungal growth (20), then the contaminated rice was dried and ground, then mixed with diet according to the Al-Shawabkeh et al. (21).

Experimental design
The birds were randomly divided into 3 groups, each subdivided into three replicates, 5 birds /replicate. First treatment: Basal starter diet. Second treatment: Basal diet with 70 ppm AFB1. Third treatment: Basal diet with 70 ppm AFB1 with 150 ppm silver nanoparticles (19).

Quantitative estimation of aflatoxin
ELIZA technique has been done to detect the AFB1 concentration in the ratio according to Al-Sawaf and Abdullah (22), Al-Dabbagh (23).

Histological analysis
The cervical displacement method was used to sacrifice the anesthetized broilers, and sections of the liver were fixed in 10% formalin for histological analysis (24).

I See Inside (ISI) technique
This method depends on the analysis of histopathological alteration in the liver according to Domingues et al. (25), who explains the ISI depends on the following evaluation: impact factor (IF), which means the histological alteration if it is reversible or irreversible, it is range from1-3, and each lesion take the score as in the table 2. ISI = IF× Score value ( Table 2).

Statistical analysis
The liver histopathological section in the variable group was analyzed. Shapiro-Wilk Test was first used for data normality, followed by Kruskal -Wali's test at P≤ 0.05.

Quantitative estimation of aflatoxin
The statistical analysis of the results of this experiment revealed there were significant differences in the concentration of aflatoxin in the experimental diets, as the significant increase of aflatoxins in the diet of the second group to which the toxins were added at a concentration of 70 parts per million and the average was 1.91 parts per gram, in contrast, to control (first) group, in the third group in which the silver nanoparticles was added to ration at 150 ppm, there was decline significantly in the aflatoxin concentration to the 1.85 ppm (Figure 1). Figure 1: Effects of Silver nanoparticles on the Aflatoxin concentration (ppm) in the broiler ratio.

The microscopic examination
The histopathological examination for the broiler liver treated with aflatoxin 70 ppm for 21 days shows variable alteration characterization by recent thrombus and proteinaceous deposition in the central vein and congestion. Dilatation in the sinusoid (Figure 2) with hepatic necrosis (Figure 3), the hyperplasia of epithelial cells lining the bile duct and sloughing to the lumen (Figure 4). Furthermore, the edema and hemorrhage are one of the hepatic circulatory disturbances and severe necrosis in the hepatic tissue ( Figure   5). The silver nanoparticles interfere with the pathological effects of aflatoxin in liver architecture, so a microscopic examination of liver feeding with a diet treated with aflatoxin and 150 ppm of silver nanoparticles revealed congestion in the hepatic central vein ( Figure 6) and dilatation of sinusoids with slight edema (Figure 7).

ISI Technique
The statistical analysis for the result of ISI of the liver section at variable groups revealed significantly elevated histopathological alteration in the liver of chicks treated with aflatoxin, but the role of silver nanoparticle was clearly in improvement in the lesions in group third in contrast to the second group but remain significantly elevated in comparison to the second group (Table 3).

Discussion
Aflatoxin is one of the main mycotoxins that cause acute or chronic toxicity, mainly affecting the liver and leading to hepatitis and carcinogenic immunosuppression for humans and animals (26,27). There are several methods (chemical and physical) for removal and detoxification the aflatoxin from feedstuff, but the green synthesis nanomaterials depend on the herbal plant extract is the recent attention from researchers and future studies mainly silver nanoparticles because it is unique characteristic as an anti-microbial agent without causing deleterious effect for human also have the ability as antifungal Candida sp. and Trichosporon (28,29). These studies revealed the activity of silver nanoparticles as an antifungal and a significant decline of the mean concentration of aflatoxin in the broiler ratio in contrast to the ratio with aflatoxin 1.86 and 1.91 ppm, respectively. These results come with the conclusion of the study of Mousavi and Pourtalebi (30), who reported that silver nanoparticles act as an antifungal and are useful to control aflatoxin contamination, Zhao et al. (31) referred to the ability of silver nanoparticles to inhibit the growth of Aspergillus flavus and decrease the production of aflatoxins.
Antifungal activity of silver nanoparticles depends on its ability to cause oxidative stress. The production of the active oxygen radical ( -O 2 ) then changes the shape and function of the fungal hyphae, or it may be the active substances that In the manufacture of silver nanoparticles in the green industry method, it is important in the absorption of aflatoxins, low absorption and rapid excretion from the gastrointestinal tract (31), the previous study has indicated that nanoparticles consider cytotoxicity cause lipid peroxidation and interact with both nucleic acid synthesis and mitochondrial functions (32,33). Silver nanoparticles are synthesized by (pomegranate peel extract). These eco-friendly substances have bioactive compound as tannin at 9.33% and polyphenolic-containing and astringent agents, which are considered toxic for organisms or may relate to physical activity between the negative charge for these substances with a positive charge for fungal spores. These lead to the inhibition of fungal growth and a decline in mycotoxin production (34).
The liver is one of the main target organs for aflatoxicosis and causes microscopic alteration (35), the histopathological investigation of this study revealed variable alteration in the hepatic architecture as congestion, recent thrombus in the central vein with edema, dilatation of the sinusoid and sever necrosis, these results come with the previous studies Magnoli et al. (36), Zabiulla et al. (37) and Ashry et al. (38), treated chick with 70 ppm of aflatoxin for 21 days lead to hyperplasia of epithelial cells lining bile ducts with sloughing in the lumen this lesion consider pathognomic lesion for aflatoxicosis, result from direct toxicity of mycotoxin or excessive production of prostaglandins (39), aflatoxin impaired lipid, protein and carbohydrate metabolism, increase oxidative stress, lipid peroxidation and disrupting cell permeability and loss ion osmoregulation all these lead to cell injury (40).
Recent studies indicated that biological and herbal plant extracts could be useful for the elimination of mycotoxin as probiotics, curcumin, eggplant shell, pomegranate extract, and sugar apple (41,42), nanomaterial as silver nanoparticles based on herbal plant extract is one of the adsorbents for aflatoxin, the improvement and protective roles of silver nanoparticles which are investigated in this study agreement with the result of Al-zubaidi et al. (43), Ismail et al. (44) and Śliżewska et al. (41) due to it is the activity for binding to the aflatoxin and increase stability it in the fluids of gastrointestinal, as it might decrease or not be absorbed via the digestive tract, another major advantage of silver nanoparticles based on green synthesis is act antifungal and inhibition fungal growth and then decrease mycotoxin production. The ISI technique has demonstrated the ability of aflatoxin to produce histopathological lesions in variable parts of the liver, which has been used by Domingues (25).

Conclusion
This study concludes that silver nanoparticles can act as an adsorbent material for aflatoxin and protect and improve tissue architecture. This study is one of the first studies using the ISI technique for determining the significant variation in the liver tissue.