Efficacy of Bentonite Nanoparticles against Necrotic Enteritis in Broiler Chickens

Nanotechnology is commonly characterized by dimensions ranging from approximately 1 to 100 nanometers (nm). This classification is grounded in the fundamental unit of measurement, the nanometer, denoted as Nano (109 meters), which is 1,000,000,000 times smaller than a meter [1]. 

The essence of nanotechnology is rooted in its application to address unresolved scientific inquiries in the realms of chemistry, physics, biology, and medicine. Scholars have formally conceptualized nanotechnology concerning nanometer-scale measurements [2-4]. While the utilization of nanotechnology in the aforementioned scientific domains remains relatively constrained, recent developments underscore its potential as an advanced tool for elucidating enigmatic phenomena and addressing unresolved issues in animal production and health [5]. 

The poultry industry is one of the most important pillars of food security in world. This sector provides achievable and affordable animal protein. It is considered the main source of protein all over the world [6]. It is well known that this sector faces many problems mainly those related to infectious diseases. Infectious diseases of poultry are generally controlled by vaccines and treated by antibiotics. 

Growing demand for poultry meat production forced producers to enhance birds’ growth at maximum rates, which can be achieved by the prevention of diseases and enhancement of growth by using sub-therapist doses of antibiotics used as growth promoters (AGP) [7]. Antibiotics Residues negatively affect human health and develop bacterial resistance [8]. These concerns have led to prohibition of use of antibiotics or AGP in animals’ feed in Jordan (the Jordanian Standards JS 7/2014, and the Ministry of Agriculture 10/1/7860/2020) and many other countries of the world. 

Necrotic enteritis (NE) is a bacterial infection disease of poultry caused by gram-positive bacteria, genus Clostridium perfringens, (types A and C), characterized by patches of necrotic tissue on the intestinal epithelium [9]. NE disease in broiler chickens usually appears between the ages of 2 and 5 weeks. C. perfringens is divided into five toxinotypes (A, B, C, D, and E), based on the presence or absence of four major toxin genes (alpha-, beta-, epsilon- and iota-toxins) in chickens, necrotic enteritis is caused mainly by type A strains [10, 11]. However, NE is an enteric disease of poultry caused by C. perfringens, gram-positive bacteria, (types A and C), characterized by patches of necrotic tissue on the intestinal epithelium [9, 12]. In a healthy bird C. perfringens population is almost less than 10⁵ colony-forming units (CFU) / g digesta. Therfore, increased number of C. perfringens more than 10⁵ cuf / g intestinal content infection of NE occurens [13]. 

Necrotic enteritis (NE) caused by C. perfringens is one of the most occurring intestinal diseases which causes severe loss in the poultry industry [14]. Due to which, economic losses occur such as high mortalities, treatment costs, heavy use of antibiotics, and a sharp reduction in weight gains and market value of the affected poultry [15]. The annual NE related economic losses have increased from two to six billion United States Dollars (USD) in your globally because it is difficult to diagnose and control disease at the early stage of occurrence [16-19]. 

Several risk factors for NE have been identified, including stress, because stress alters the intestinal environment and increases the risk of induction, it could be a risk factor for NE in broiler chickens [20]. The occurrence and severity of NE lesions are increased by heat stress. Heat stress significantly increases the number of NE-positive birds and the severity of NE lesions in challenged birds [21]. However, coccidiosis is the most common risk factor, and it is caused by coccidial pathogens Eimeria spp that damage the intestinal mucosa [22, 23] reported that the gut microbiota and immune system of the bird can also be significantly altered by Eimeria infection, which increases the likelihood of C. perfringens colonization. Coccidiosis is as define a parasitic chicken's broiler disease, which is caused by Eimeria spp. and causes losses to the global economy [24]. 

NE and Coccidiosis are enteric diseases caused by the bacterial pathogen Clostridium perfringens and the coccidial protozoan genus Eimeria. Coccidiosis is a major parasitic disease caused by Eimeria (E) protozoa. E. acervulina, E. maxima, and E. tenella are the species that affect broiler chickens. These species cause typical lesions in the duodenum, jejunum, and cecum, respectively, and have the highest occurrence in broiler flocks, which are commonly monitored; also Coccidiosis plays an important role in the development of NE [25]. 

Elevated levels of indigestible, water-soluble, non-starch polysaccharides in the diet increase the incidence of NE. Wheat, barley, and rye for example, are risk factors for NE, whereas corn is not [26-28]. High levels of animal protein in the diet, such as fishmeal, have also been linked to an increase in the occurrence of NE [29]. The dietary fat source may also have an impact on the count of C. perfringens. When compared to vegetable oil, animal fat increases C perfringens counts [30, 31] reported that higher stocking density reduces bird welfare, as evidenced by lower production, poorer litter quality, and increases in footpad lesions, infectious mortality, fear, and stress. Impacts of housing densities on Gastro Intestinal Tract (GIT) environment in negativaly by increasing the risk of NE occurrence, also the rise in the number of birds per unit area causes stress in the birds, which eventually leads to NE [32]. Tsiouris [21] showed that heat stress has a negative impact on NE and gut health. However, the use of AGP was also considered responsible for increased the spread of NE disease [33]. 

In poultry, antibiotics such as tylosin, bacitracin, lincomycin, penicillin, and tetracycline are commonly used for the prevention and treatment management of C. perfringens-induced neonatal diarrhea and NE [34]. Antibiotics are the primary treatment and control strategy for necrotic enteritis in broiler chickens [35]. 

Bentonite is a natural clay material, abundant in Jordan, has low cost, safe, and convenient for use in broiler chickens [36]. The antibacterial properties of bentonite have been confirmed [37]. The term "bentonite" refers to the clay mineral montmorillonite in its practical form [38]. Most bentonite deposits are formed as a result of volcanic ash alteration, primarily in damp or wet conditions, or as a result of primary rock decomposition in water [39]. One of the primary characteristics of bentonite clay is its ability to absorb water. The degree of hydration and swelling that result is determined by the kind of exchangeable ions that are present, each having varied hydrophilic and solvating qualities [40]. Bentonite clay powder is a good stabilizing agent because it fills the voids between sand particles and reduces the amount of free water in the voids, increasing sand mass strength [41]. 

The nanoparticle dosage form of bentonite is a new innovative application in this field, due to their healing and antimicrobial properties bentonites are vastly used in the pharma, cosmetics and agriculture industry [42]. Their antibacterial activity came from their remarkable high cation exchange capacity, high surface area, high swelling capacity, high water dispersibility, high absorption capacity, and non-toxic properties [37]. In addition, the nanoparticles of bentonites have large surface areas which increase their ability to absorb toxins produced by microorganisms. 

Finding alternative therapy to antibiotics is considered challenging and of high importance. In this study, NE was induced by infecting broilers with coccidia and C. perfringens, while the nanoparticles of bentonite were evaluated as an alternative to antibiotic treatment. The availability of bentonite in world in huge amounts. Moreover, bentonite has no negative effects on human health and it might resolve the problem of antibiotic residues in poultry products. Therefore, the objective of this study was to investigate the impact of bentonite nanoparticles on the productive performance, carcass characteristics, intestinal morphology and oocyte shedding in broiler chickens infected with C. perfringens compared with the control of broiler chickens.

Birds and housing, Experimental Design 

This study was conducted at the poultry farm belonging to the University of Jordan in Almuwaqar research station. A total of 300 one-day-old broiler chicks (Ross 308) were provided by a commercial hatchery. The chicks were randomly allocated into 5 treatments in a completely randomized design (CRD) with three replicates (pens) per treatment and 20 birds per replicate (60/treatment). Management practices were in accordance with the standard guidelines provided by the strain management guide. Feed and water were provid. The experiment was conducted for 28 days. The rearing period was divided into 2 phases: starter diet (1-14 days) and grower diet (15-28 days). All treatments received the same diets throughout the study. The diets composition is shown in (Table 1). But, the diet in this experiment was antibiotic and coccidostat-free. 

Table 1: Composition and Calculated Analysis of the Basal Diets Fed During Starting (1 - 14) and Growing Periods (15 - 28) Days of Age.

Treatments 

Experiment was consisted of the following 5 treatments (T) 

• T1: Non Infected + No Treated (Negative Control).
• T2: Infected + Non Treated (Positive Control).
• T3: Infected + Antibiotic Treated (Antibiotic Groupe).
• T4: Infected + ASBN Treated (2% ASBN).
• T5: Infected + ASBN Treated (4% ASBN).

Method of administration

The antibiotic employed in this study was "Lincomycin™," administered at a dose in accordance with the manufacturer's recommendations, specifically 17 mg of Lincomycin per 1000 ml of drinking water. Treatment T3 involved the administration of the antibiotic for three consecutive days, following the manufacturer's guidelines. In contrast, ASBN was provided once per week for a 4-hour duration in the designated treatments, adhering to the protocol established in Experiment 1. 

To initiate the administration of the antibiotic, ASBN, or tap water, bell drinkers were removed from all treatments for a 2-hour interval. The requisite doses of ASBN (T4 and T5) were dispensed in water for a 4-hour duration, with the water intake adjusted based on the age of the birds. Concurrently, T1 and T2 were provided with plain tap water. All birds had unrestricted access to water, with additional drinkers available to ensure ample water accessibility. The quantity of consumed water was determined in accordance with the guidelines outlined in the strain manual, ensuring that the provision exceeded the specified requirements. Water consumption was recorded for each pen, and after 4 hours, the regular tap water was reinstated to the chicks. This procedural regimen was reiterated on a weekly basis throughout the 4- week trial period. 

Method of challenging the birds 

Necrotic enteritis (NE) was induced by infecting the birds with sporulated Eimeria spp. and a pathogenic strain of C. perfringens (Cp). NE was achieved through 2 successive steps. First, by infecting birds with sporulated Eimeria spp. to induce coccidiosis. A coccidiosis infection was induced before the C. perfringens infection take place to provide a predisposing condition for NE. Coccidiosis infection was induced by inoculating the birds at 7-days of age with 25x the recommended dose of coccidia vaccine (COCCIVACTM-D2®; Intervet Inc., Omaha). The COCCIVACTM-D2® was used to prepare an inoculum composed of live oocytes of 6 Eimeria spp.; namely Eimeria acervulina, E. brunetti, E. maxima, E. mivati, E. necatrix and E. tenella. The inoculum was prepared to provide 25 times the recommended dose of the vaccine for birds. This protocol was proven to induce coccidia infection and intestinal mucosal damage in chickens (Unpublished data, Personnel communication with Dr. Abdelqader, the University of Jordan). A number of live oocytes in the inoculum were counted using a McMaster counting slide under the microscope to calculate the number of live oocysts per ml. Birds were infected by oral gavage directly into the crop, while birds assigned to non-challenged group was gavaged with distilled water using the same volume calculated for the challenged birds. 

The second step of the NE challenge is to infect the birds at the age of 14 days (7 days post coccidian infection) with a pathogenic strain of C. perfringens. The Clostridium perfringens pathogenic strain was provided by Dr. Mohammad Gharaibeh, Faculty of Veterinary Medicine, Jordan University of Science and Technology, and prepared by Dr. Gharaibeh to be ready for oral infection of chickens. The Clostridium perfringens pathogenic strain was isolated and identified out of 60 Clostridium perfringens strains isolated from Jordan chickens’ farms and identified by using appropriate gene sequencing procedures. 

Bentonite Nanoparticles Preparation 

Bentonite sourced from Al-Azraq region in Jordan. The transformation of bentonite into nanoparticles occurred at the Jordan University of Science and Technology (JUST) – Nano Institute. Nano-sized particles were generated using a ball milling machine, specifically the Planetary Micro Mill (Model: PULVERISETTE 7, Manufacturer: Fritsch, Country: Germany). The ball milling process was executed using this instrument located at the JUST – Nano Institute Lab. The jar was filled with powdered samples, and balls of appropriate size and quantity, commensurate with the powder in terms of hardness, were added. The milling program was configured at 800 rpm, 20 cycles, and 3 minutes for each cycle. The resultant bentonite nanoparticles achieved a size reduction to 100 nm. Verification of the particle size of bentonite was conducted using a scanning electron microscope (SEM image 600,000X-200 nm), as depicted in (Figure 1). The SEM instrument used in this analysis was the QUANTA FEG 450 (Manufacturer: Thermo Fisher Scientific, Country: America), available at the JUST – Nano Institute Lab.

 Figure 1: Image of Bentonite Nano at Scanning Electron Microscope (SEM).

SE mode, image 600.000X - 200 nm 

In this experimental endeavor, bentonite nanoparticles were combined with drinking water to formulate solutions with varying concentrations. The chemical composition of raw bentonite was determined through X-Ray Fluorescence analysis by the Natural Resources Authority Directorate in Amman, Jordan, and is presented in Table (2).

Table 2: Chemical Composition of Bentonite in Raw Form

Provided by the Natural Resources Authority/ Laboratories Directorate, X-Ray Fluorescence Analysis. Amman-Jordan. Loss of Ignition (LOI)

Performance Parameters 

Average feed intake (FI), body weight (BW), and feed conversion ratio (FCR) were recorded on a weekly base. The offered feed and their refusals were recorded daily and at the end of each week. Feed refusals were weighed and subtracted from the offered to calculate total feed consumption during the week. The weekly pen feed consumption value was then divided by the number of birds/pen after adjustment for mortality to obtain the adjusted average feed consumption per bird throughout the week. 

Body weight gain and FCR was also calculated on weekly basis through the experimental period as shown below: 

• BWG = Final weight – Initial weight / Number of days.
• FCR = Feed consumed in grams / Body weight gain in grams.

Carcass characteristics 

At the end of the experiment, 6 birds were randomly selected from each treatment. Birds were slaughtering and directly taken to perform the following measurements: hot carcass weight, breast meat yield, and weight of the liver, weight of the heart, weight of the gizzard, and weight of the spleen. The dressing % was calculated as the relation of hot carcass weight to live weight (g/g ×100). The relative weight of internal organs was calculated as a ratio of an organ weight divided by the birds live weight (g/kg of body weight).

Intestinal histology 

At the end of the experiment, the same birds euthanized above for carcass quality were used for intestinal morphology analysis. The preparations of intestinal tissues for histology measurements were performed. Concisely, the duodenum section was used to cut a segment of 5 cm in length and placed buffered 10% formalin, processed in alcohol of different concentrations using (Histokinette device 2000) for fixation and dehydration. Then dehydrated tissues were covered with paraffin at 45 C using paraffin dispenser. Then paraffin cassettes allowed to cool at room temperature, they and stored at freezing temperature until the time of cutting and slides preparation. Paraffin cassettes were cutted using Microtome device at a thickness of 5 μm. Multiple slides were prepared from each tissue. Then, all slides were stained using Haematoxylin and Eosin (HE) stain (two jars then dehydration using alcohol 70% then 80% then two jars of 95 and two jars of 100%, then clearing using two changes of xylene, then infiltration using two changes of parrafin wax, then embedding. Stained slides were viewed under a compound microscope to measure villi morphometric measures in the duodenum using specific computer software (Motic image 2.0 multi- language) to capture images of the tissues. Images were used to measure villi length, crypt depth and villus width of different treatments. Stained slides were viewed under compound microscope to record villi morphometric measures. Also, villi length, crypt depth and villus width of the different treatments. 

Villus surface area: calculated using the formula: villus area = (2π) × (VW/2) × (VH) in which VW = villus width and VH = villus height.

Intestinal lesion scoring

At the end of the trial, the same birds (6/treatment) used for carcass quality analysis were used for intestinal lesion scoring. After collecting the histology samples, the whole intestines were opened longitudinally and scored on a scale of 0 to 4 for lesions of damage at three sites: the duodenum (from the pyloric junction to the most distal point of insertion of the duodenal mesentery), upper half of the jejunum, and lower half of the jejunum. The scores are adopted according to the method described by Johnson and Reid [43]. A score of zero represents absence of gross lesions and 4 represented extensive hemorrhage or lesions. The lesion scores were recorded as the average across the birds at each segment. Total lesion score was calculated as the sum of lesion scores in the three intestinal segments (duodenum, upper jejunum, and lower jejunum). The lesion scoring system was: 0 = No gross lesions. l =Small scattered petechiae and white spots easily seen from the serosal side; little if any damage apparent on the mucosal surface. 2 = numerous petechiae on the serosal surface; slight ballooning confined to the midgut area may be present. 3 = Extensive hemorrhage into the lumen of the intestine; serosal surface is covered with red petechiae and/or white plaques. The serosal surface is rough and thickened with many pinpoint hemorrhages. Normal intestinal contents are lacking; ballooning extends over lower half of small intestine. 4= Extensive hemorrhage giving the intestine a dark color; intestinal contents consist of red or brown mucus. Ballooning may extend throughout much of the length of the intestine. Dead birds are scored as 4.

Oocyst excreta shedding 

Excreta samples were collected from all treatments, from each replicate, starting on day one before inducing the infection and at days 14 and 21of bird's age. Excreta samples were examined for coccidia oocysts by a modified McMaster technique and flotation method in a saturated sodium chloride solution. 

Four grams of faeces were weighed and placed into a container with 56 ml of flotation fluid (400 grams of sodium chloride dissolved in 1000 ml of distilled water). The solution was vigorously mixed before it was filtrated. The solution was loaded onto a McMaster Egg Slide for oocyst counting and examined under a compound microscope at 10 x 10 magnifications. The number of oocysts per gram (OPG) of faeces is calculated as the number of oocysts in two chambers multiplied by 50. 

Statistical Analysis 

The Statistical Analysis System (SAS Institute, 2010, Version 9.1.3) was used to conduct all statistical analysis. For performance data, the replicate was considered as the experimental unit, and analyzed with one-way ANOVA implemented using GLM procedure of SAS. For carcass, intestinal lesion, and histological data the bird was considered as the experimental unit. Differences were accepted as representing statistically significant differences when P≤0.05. Duncan multiple range test was used to separate significant means. 

Body weight, weight gain, feed Intake and feed conversation ratio 

There was no significant difference (P? 0.05) between the treatments in body weight (BW) at the age of 7 and 14 days old. Results showed a significant difference in bird body weight between the treatments at day 28 of the rearing periods. On day 28 the highest (P< 0.05) body weight was for T5 (4% ASBN) which was (1702.13g), as shown in Table (3).

Table 3: Effect of Feeding Different Levels of ASBN on Production Performance of Broiler Chickens infected with NE

a, b related to the significant differences between the treatments on p≤0.05.

*SEM: standard error of the mean

T1: Non Infected + No Treated (Negative Control), T2: Infected + Non Treated (Positive Control), T3: Infected + Antibiotic Treated (Antibiotic Groupe), T4: Infected + 2% ASBN Treated (2% ASBN), T5: Infected + 4% ASBN Treated (4% ASBN).

There was no significant difference (P? 0.05) between the treatments in body weight gain (BWG) at age 14 days old. The results showed a significant difference (P< 0.05) in bird body weight gain (BWG) between the treatments on days 28, of the rearing periods. On day 28 the highest significant body weight gain was for T5 (4% ASBN) which was (1185.53g) and total body weight gain (1662.60g). On the other hand, there were no significant differences between treatment groups on feed intake, and feed conversation ratio (FCR) of the rearing as shown in (Table 7).

Table 4: Effect of ASBN on Intestinal Histology (Duodenum) at the End of Experiment (28 Day Old)

Mortality rate

Daily mortalities were recorded for each replicate within treatment, and then the overall mortality rate was calculated as the number of dead chicks subtracted from the number of live chicks at the beginning of that week is presented in (Table 3). The mortality rate was not significantly lowered in all ASBN treated groups and antibiotic group compared with the negative control group and positive groups. 

Carcass characteristics 

Carcass characteristics parameters (dressing and cut percentages) are presented in (Table 4). The values of the dressing, lever, heart, spleen, and gizzard were not significantly affected by treatments.

Table 5: Effect of Feeding Different Levels of ASBN on Dressing Percentage and Carcass Cuts as a Percentage to Carcass Weight.

Means with the same letter in the same raw are not significantly different at p≤0.05 / * SEM: standard error of the mean

T1: Non Infected + No Treated (Negative Control), T2: Infected + Non Treated (Positive Control), T3: Infected + Antibiotic Treated (Antibiotic Groupe), T4: Infected + 2% ASBN Treated (2% ASBN), T5: Infected + 4% ASBN Treated (4% ASBN).

Oocyte count, Intestinal Histology and Intestinal lesion scoring:

The number of fecal oocyte shedding in each treatment is presented in (Table 6). According to these results, T2 (positive control) showed a significantly higher fecal oocyte count compared with other of the treatments, followed by T3 (antibiotic group), then T1 (negative control group) and T4 (2%ASBN) and T5 (4%ASBN). 

Table 6: Number of Fecal Oocytes Count on Days 14 and 21 of birds age Infection with Coccidiosis.

Opg: oocytes per gram of faeces. *SEM: standard error of the mean.

a, b related to the significant differences between the treatments on p≤0.05.

T1: Non Infected + No Treated (Negative Control), T2: Infected + Non Treated (Positive Control), T3: Infected + Antibiotic Treated (Antibiotic Groupe), T4: Infected + 2% ASBN Treated (2% ASBN), T5: Infected + 4% ASBN Treated (4% ASBN).

Results of intestinal lesion scoring are shown in (Table 7). The positive control group showed a significant increase in the score compared with other treatments as it gave the highest rate of scoring, followed by T4 (2%ASBN) and T3 (antibiotic group), then T1 (negative control group) and T5 (4%ASBN).

Table 7: Effect of ASBN on Intestinal Lesion Scoring at the End of Experiment (28 Day Old).

*SEM: standard error of the mean.

T1: Non Infected + No Treated (Negative Control), T2: Infected + Non Treated (Positive Control), T3: Infected + Antibiotic Treated (Antibiotic Groupe), T4: Infected + 2% ASBN Treated (2% ASBN), T5: Infected + 4% ASBN Treated (4% ASBN).

The results showed a significant increase (P< 0.05) in duodenum villus width, crypt depth, height: crypt ratio, and the villus area as shown in Table (7). The differences in the villus length appear clearly in the (Figure 2). The best appearance for villus is for the 4% ASBN group and for the antibiotic group. While there was a clear reduction in the villus length in the positive control group and the group that takes 2% ASBN, and the negative control group. 

 Figure 2: Effect of ASBN on duodenum Histology at the End of Experiment (28 Day Old). 

Birds in the 4% ASBN group had significantly (P < 0.05) higher values of villus height, villus width, crypt depth and villus surface area in the duodenal segment in comparison to other treatment groups. Villus height, villus width, crypt depth and villus surface area highest in the 4% ASBN group were 1212.77 μm, 169.66 μm, 303.40 μm and 0.64703 mm² respectively in comparison to other treatment groups. The lowest significant villus length (1083.83 μm) was for the positive control group.

Body weight, weight gain, feed Intake and feed conversation ratio 

There was no significant difference (P ? 0.05) between the treatments in body weight and body weight gain at the 7 and 14 days of rearing. The concentration and dose of ASBN used in this study may have contributed to the effects of ASBN on body weight gain and body weight. Moreover, there were significant differences at (P < 0.05) between treatment groups in bird on body weight gain and body weight of the rearing periods, on day 28 the highest significant weight gain was for T5 (4% ASBN). This result suggests that it is possible to recommend giving it more than once a week in order to achieve a quick result. This result was similar to Ghazalah [44] who noted the improved body weight gain with the addition of bentonite 0.50% for alonger time during 29–35 days compared to other treatments. Moreover, the results are consistent with the finding of Hamouni [45] who showed a significant improvement in weight gain of bentonite sodium 5% supplementation in for chickens compared to the control. Furthermore, Salari [46] reported that the chickens fed containing 1-2 % sodium bentonite consumed more feed, and had more weight gain. 

The results revealed that there was no significant difference in feed intake and feed conversation ratio between the treatment groups in the rearing period after the second week and lasted until the end of the rearing period. This is explained by the fact that BW and BWG were not affected at the rearing period, the number of times ASBN was added and the challenges infected birds with sporulated Eimeria spp and C.perfringens. Our results varied from Al-Beitawi [47] who found that the use of a 2% concentration of the Aqueous Nanosuspension of clay minerals improved the growth performance of broiler chickens in terms of the FCR. Our results were comparable to Karomy and Maged [48] who showed that the used 1, 2% sodium bentonite, and 1% sodium bentonite with 1% aluminum silicate can be used in pelleted making diets for improving growth performance, decreased feed intake and enhance feed efficiency ratio. On the other hand, Ahmadi [49] reported that the use of Silver Nanoparticles 900ppm in broiler chickens improved growth performance in terms of body weight, feed intake and feed conversion ratio. Jarocka [50] found that the supplemented of 2.2 mg/ml gold nanoparticles improved the growth performance of poultry birds. 

Carcass characteristics 

There were no significant differences between carcass characteristics parameters dressing, lever, heart, spleen, and gizzard in birds between all of the treatment groups, the reduction in the feed intake was reflected on carcass weight parts and dressing percentage. Our results varied from Al-Beitawi [51] who reported that the 1.5% nano clay minerals fed improved broiler internal organs measurements. Moreover, Al-Beitawi [47] reported that the use of 1.5 % and 2 % of aqueous nano suspension of clay minerals to broiler increased liver, gizzard, heart, pancreas and spleen percentage. Furthermore, Al-Beitawi [51] and Al-faqieh [36] carried out their experiments under normal conditions, in contrast to the present experiment where the birds were infectd with C. Perfringens. 

Intestinal Histology Intestinal lesion scoring and Oocytes count 

There were significant differences in villus width, crypt depth and villus surface area in the duodenal segment, the highest significant percentage was seen in the T5 (4% ASBN) group than in other treatment groups. Khan [52] showed that bentonite bound 90% of the aflatoxins present in feed and made them effective and unabsorbable from the gastrointestinal tract. Furthermore, it has been noted that the characteristics of nanoparticles increase their appeal for enhancing the bioavailability and absorption of extra nutritional substances like vitamins and minerals [10]. These results attributed to the beneficial properties of nano bentonite: high capacity for absorption, a large surface area, improvement in the enzymatic activity of the digestive tract, and lowering the viscosity of fluids. The results of this study correspond with those reported by Besseboua [53] who noted that the diet supplemented with bentonite 4% dose increased the villus height of jejunum in broiler. Ahmadi [54] reported that 0.4 mg/kg nano selenium supplementation in the diet from (1-42) days of age has a positive effect on the duodenum, jejunum, and ileum, resulting in an increase in length, which is directly related to better resorption. The supplementation of 100 mg/kg copper nanoparticles feed improved immunity, protein synthesis and caecal microbiota in broilers [55]. Karomy and Maged [48] showed that the supplanted 1 and 2 % sodium bentonite improved ameliorated Protein digestibility, protein efficiency, and viscosity in broiler chickens. 

The results of oocyte counting, T2 (Positive Control) showed a significant difference with other treatments as it gave the highest, followed by T3 (Antibiotic Group), then Negative Control (T1), 2% ASBN (T4) and 4% ASBN (T5) at days 7 and 14 after induction of coccidiosis. In the current study, supplementation of ASBN may protect against Eimeria spp infection in the broiler. However, the results of intestinal lesion scoring in positive control showed a significant difference with other treatments as it gave the highest rate of scoring, followed by 2% ASBN, antibiotic group, then negative control and 4% ASBN. This would be also attributed to the ability of ASBN to bind with the pathogenic microbes that may be established in the gut of broiler chickens. This result was similar to Xu [56] who reported that the use of dietary supplementation of newly hatched broiler chicks with 0.25mg/kg sodium selenite from the day of hatch significantly reduced NE-induced gut lesions. These results disagreed with Hayajneh [57] who reported that the use of 6% calcium bentonite in the diet of broiler chickens did not showe any significant number of oocysts counts, which were the highest in the negative control group followed by the bentonite group than positive control group (Amprolium). It is critical to monitor C. perfringens infections on a regular basis to avoid the development of antibiotic resistance. Indeed, Particles with nano-diameter absorb faster through diffusion from gut mucus to reach the intestinal lining cells and blood. This would be due to the significance of nano bentonite's beneficial properties such as the high capacity for absorption, the ability of nano particles to bind with pathogens, a large surface area, improvement in the enzymatic activity of the digestive tract, and increasing the pH and lowering the viscosity of fluids.

The experiment utilization of an aqueous suspension of bentonite nanoparticles as a substitute for antibiotics in combating Necrotic Enteritis presents a promising avenue for enhancing both the performance and health of chickens under the NE challenage. The administration of 2% and 4% ASBN in drinking water, once weekly to NE infected broiler chickens, manifested positive effects including increased body weight gain, decreased mortality rates, reduced intestinal lesion scoring, diminished oocyte count, and enhanced the duodenal segment morphometrics such as villus length, villus width, crypt depth, and villus surface area. These alterations collectively contribute to an integrated intestinal surface area with the potential to enhance digestion and absorption. 

Under the NE challenge, the use of 2% ASBN would be a safe alternative to antibiotics. This transition not only addresses concerns about antibiotic residues in meat and the development of microbial resistance, while further studies should invesitgat the effect of ASBN under other infections and different challenges.

I would like to thank for H. E. Dr. Khalifa Musabeh Alteneiji, my colleagues DAL/ UAE and Eng. Razan Fahad Alhammadi. I would like to express my sincerely to Prof. Anas Abdelqader, Prof. Talal Aburjai and Dr. Mohammad Gharaibeh for their invaluable guidance and advice, then special thanks to  The University of Jordan, JUST, NCRD, DAL, EKTIFA, and my colleagues / NCRD  for their help in my research.

1. Dera MW, Teseme WB, Mulugeta Wegari Dera WBT (2020) Review on the application of food nanotechnology in food processing. American Journal of Engineering and Technology Management 5: 41-47.
2. Ramirez-Mella M, Hernandez-Mendo O (2010) Nanotechnolgy on animal Tropical and Subtropical Agroecosystems 12: 423-429.
3. Buzea C, Pacheco II, Robbie K (2007) Nanomaterials and nanoparticles: sources and Biointerphases 2: 17-71.
4. Scott NR (2005) “Nanotechnology and Animal ” Rev Sci Tech Off Int Epiz 24: 425-432.
5. Patil SS, Kore KB, Kumar P (2009) Nanotechnology and its applications in veterinary and animal Vet World 2: 475-477.
6. Mottet A, Tempio G (2017) Global poultry production: current state and future outlook and World’s Poultry Science Journal 73: 245-256.
7. Broom LJ (2017) The sub-inhibitory theory for antibiotic growth Poultry science 96: 3104-3108.
8. Berghiche A, Khenenou T, Labiad I (2019) A Meta-Analysis on antibiotic residues in meat of broiler chickens in developing Journal of World’s Poultry Research 9: 89-97.
9. Timbermont L, Haesebrouck F, Ducatelle R, Van Immerseel F (2011) Necrotic enteritis in broilers: an updated review on the pathogenesis. Avian Pathology 40: 341-347.
10. Rood JI, Adams V, Lacey J, Lyras D, McClane BA, et al. (2018) Expansion of the Clostridium perfringens toxin- based typing scheme. Anaerobe 53: 5-10.
11. Petit L, Gibert M, Popoff MR (1999) Clostridium perfringens: toxinotype and Trends in microbiology 7: 104-110.
12. Lacey JA, Johanesen PA, Lyras D, Moore RJ (2016) Genomic diversity of necrotic enteritis-associated strains of Clostridium perfringens: a Avian Pathology 45: 302-307.
13. Kondo F (1988) In vitro lecithinase activity and sensitivity to 22 antimicrobial agents of Clostridium perfringens isolated from necrotic enteritis of broiler chickens. Research in veterinary science 45: 337-340.
14. Aytek E, Adiguzel MC (2023) Alternative Strategies for Control of Necrotic Enteritis in Poultry. Innovative Research in Health Sciences 105-124.
15. García-Vela S, Martínez-Sancho A, Said LB, Torres C, Fliss I (2023) Pathogenicity and Antibiotic Resistance Diversity in Clostridium perfringens Isolates from Poultry Affected by Necrotic Enteritis in Pathogens 12: 905.
16. Van der Sluis W (2000a) Clostridial enteritis a syndrome emerging world World Poultry 16: 5657.
17. Van der Sluis W (2000b) Clostridial enteritis is an often underestimated World Poultry 16: 4243.
18. Wade B, Keyburn A (2015) The true cost of necrotic enteritis. World Poultry 31:16-17.
19. Zahoor I, Ghayas A, Basheer A (2018) Genetics and genomics of susceptibility and immune response to necrotic enteritis in chicken: a Molecular biology reports 45: 31-37.
20. McDevitt RM, Brooker JD, Acamovic T, Sparks NHC (2006) Necrotic enteritis; a continuing challenge for the poultry industry. World’s Poultry Science Journal 62: 221247.
21. Tsiouris V, Georgopoulou I, Batzios C, Pappaioannou N, Ducatelle R, et al. (2018) Heat stress as a predisposing factor for necrotic enteritis in broiler chicks. Avian Pathology 47: 616-624.
22. Williams RB (2005) Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut Avian Pathology 34: 159180.
23. Rodgers NJ, Swick RA, Geier MS, Moore RJ, Choct M, et al. (2015) Multifactorial analysis of the extent to which Eimeria and fishmeal predispose broiler chickens to necrotic Avian Dis 59: 38-45.
24. Nowaczewski S, Janiszewski S, Kaczmarek S, Kaczor N, Racewicz P, et al. (2023) Evaluation of the effectiveness of alternative methods for controlling coccidiosis in broiler chickens: a field Animal Science Papers and Reports 41: 97-110.
25. Nawarathne SR, Yu M, Heo JM (2021) Poultry Coccidiosis-A Concurrent Overview on Etiology, Diagnostic Practices, and Preventive Korean J. Poultry Science 48: 297-318.
26. Craven SE (2000) Colonization of the intestinal tract by Clostridium perfringens and fecal shedding in diet-stressed and unstressed broiler Poultry Science 79: 843-849.
27. Jia W, Slominski BA, Bruce HL, Blank G, Crow G, et al. (2009) Effects of diet type and enzyme addition on growth performance and gut health of broiler chickens during subclinical Clostridium perfringens Poultry science 88: 132-140.
28. Kaldhusdal M, Skjerve E (1996) Association between cereal contents in the diet and incidence of necrotic enteritis in broiler chickens in Preventive Veterinary Medicine 28: 1-16.
29. Drew MD, Syed NA, Goldade BG, Laarveld B, Van Kessel AG (2004) Effects of dietary protein source and level on intestinal populations of Clostridium perfringens in broiler chickens. Poultry science 83: 414-420.
30. Knarreborg A, Simon MA, Engberg RM, Jensen BB, Tannock GW (2002) Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages. Applied and environmental microbiology 68: 5918-5924.
31. Shynkaruk T, Long K, LeBlanc C, Schwean-Lardner K (2023) Impact of stocking density on the welfare and productivity of broiler chickens reared to 34 d of Journal of Applied Poultry Research 32: 100344.
32. Qaid M M, Albatshan HA, Hussein EO, Al-Garadi MA (2023) Effect of housing system and housing density on performance, viability, and gastrointestinal tract growth of broiler chicks during the first 2 wk of Poultry Science 102: 02752.
33. Riaz A, Umar S, Munir MT, Tariq M (2017) Replacements of antibiotics in the control of necrotic enteritis: a review. Sci Lett 5: 208-216.
34. Anju K, Karthik K, Divya V, Priyadharshini MLM, Sharma RK, et al. (2021) Toxinotyping and molecular characterization of antimicrobial resistance in Clostridium perfringens isolated from different sources of livestock and Anaerobe 67: 102298.
35. Dahiya JP, Wilkie DC, Van Kessel AG, Drew MD (2006) Potential strategies for controlling necrotic enteritis in broiler chickens in post-antibiotic Animal Feed Science and Technology 129: 60-88.
36. Al-Faqieh MA, Abdelqader A, Aburjai T (2024) Effect of Different Levels of Aqueous Suspension of Bentonite Nanoparticles on Performance and Carcass Characteristics of Broiler Jordan Journal of Agricultural Sciences 20:141-148.
37. Martsouka F, Papagiannopoulos K, Hatziantoniou S, Barlog M, Lagiopoulos G, et al. (2021) The Antimicrobial Properties of Modified Pharmaceutical Bentonite with Zinc and Pharmaceutics 13: 1190.
38. Maged A, Kharbish S, Ismael IS, Bhatnagar A (2020) Characterization of activated bentonite clay mineral and the mechanisms underlying its sorption for ciprofloxacin from aqueous Environmental Science and Pollution Research 27: 32980-32997.
39. Muhammad N, Siddiqua S (2019) Stabilization of silty sand using bentonite-magnesium-alkalinization: Mechanical, physicochemical and microstructural characterization. Appl Clay Sci 183: 05325.
40. Rios S, Viana de FA, Bangaru S (2016) Silty sand stabilized with different binders. Procedia Eng 143: 187-195.
41. Bani Baker M, Abendeh R, Sharo A, Hanna A (2022) Stabilization of sandy soils by bentonite clay slurry at laboratory bench and pilot Coatings 12: 1922.
42. Srasra E, Bekri-Abbes I (2020) Bentonite clays for therapeutic purposes and biomaterial design. Current Pharmaceutical Design 26: 642-649.
43. Johnson J, WM Reid (1970) Anticoccidial drugs: Lesion scor-ing techniques in battery and floor pen experiments with Exp Parasitol 28: 30-36.
44. Ghazalah AA, Abd-Elsamee MO, Moustafa KEM, Khattab MA, Rehan AEA (2021) Effect of Nanosilica and Bentonite as Mycotoxins Adsorbent Agent in Broiler Chickens’ Diet on Growth Performance and Hepatic Animals 11: 2129.
45. Hamouni R, Mimoune N, Benaissa MH, Baazizi R, Kaidi R, et al. (2020) Effect of different levels of bentonite supplementation in diets on zootechnical performance of broiler chicken. Agricultura 115: 3-4.
46. Salari S, Kermanshahi H, Moghaddam HN (2006) Effect of sodium bentonite and comparison of pellet mash on performance of broiler chickens. International Journal of Poultry Science 5: 31-34.
47. Al-Beitawi N, Elshuraydeh K, Al-Faqeih M, Zyadeh M (2017a) Effect of an Aqueous Nanosuspension of Clay Minerals on the Performance, Carcass Characteristics and Internal Organs of Journal Nanotechnol Nanomed Na-nobiotechnol 4: 013.
48. Karomy QJGAS, Maged TI (2021) Effect of Feed Supplemented with Different Levels of Sodium Bentonite and Aluminum Silicate on Physiological Performance of Broiler, Indian Journal of Ecology 48: 500-505.
49. Ahmadi J (2009) Application of Different Levels of Silver Nanoparticles in Food on the Performance and Some Blood Parameters of Broiler Chickens. World Applied Sciences Journal 7: 24-27.
50. Jarocka U, Sawicka R, Góra-Sochacka A, Sirko A, Zagórski-Ostoja W, et al. (2014) An immunosensor based on antibody binding fragments attached to gold nanoparticles for the detection of peptides derived from avian influenza hemagglutinin H5. Sensors 14: 15714-15728.
51. Al-Beitawi NA, Momani Shaker M, El-Shuraydeh KN, Bláha J (2017b) Effect of nanoclay minerals on growth performance, internal organs and blood biochemistry of broiler chickens compared to vaccines and Journal of Applied Animal Research 45: 543-549.
52. Khan AD, Ijaz N, Shahzad K (2001) Role of bentonites in poultry Agro Vet News 2-3.
53. Besseboua O, Ayad A, Hornick JL, Benbarek H (2019) Dietary effects of Algerian sodium bentonite on growth performance and biochemical parameters in broiler chickens. Cercet?ri Agronomice în Moldova 2018: 108-121.
54. Ahmadi M, Ahmadian A, Poorghasemi M, Makovicky P, Seidavi A (2019) Nano-Selenium affects on duodenum, jejunum, ileum and colon characteristics in chicks: An animal model. International Journal of Nano Dimension 10: 225-229.
55. Mroczek-Sonsowska N, Lukasilewics M, Wnuk A, Sawsz E, Niemiec J (2014) Effect of copper nanoparticles and copper sulfate administered in ovo on copper content in breast muscle, liver and spleen of broiler chickens. Annals of Warsaw University of Life Sciences - SGGW. Animal Science 53: 135-142
56. Xu SZ, Lee SH, Lillehoj HS, Bravo D (2015) Dietary sodium selenite affects host intestinal and systemic immune response and disease susceptibility to necrotic enteritis in commercial broilers. British Poultry Science 56:103-112.
57. Hayajne FMF, Abdelqader A, Alnimer MA, Abedal-Majed MA, Al-Khazaleh J (2020) The role of high-grade Bentonite powder in coccidiosis and its effects on feed conversion ratio and blood parameters in broiler Polish Journal of Veterinary Sciences 23: 97-107.