Nano silver treats necrotic enteritis in broilers caused by Clostridium perfringens bacteria

A total of 120 Cobb broilers (1 day old) were collected for this study and divided into 4 equal groups at 14 days of age (30 birds each); Each group was divided into 3 equal replicates (10 animals each). The groups are designated as follows:

G1, infected;

G2, infected and treated with nano silver AgNPs;

G3, treated with AgNPs;

and G4, negative control.

Birds were infected with 4×108 colony forming units (CFU)/mL/bird of C. perfringens type A bacteria on 2 consecutive days. In the treated groups, AgNPs (average diameter 15 nm; total dose 150 µg/animal) were administered through the plant digestive tract. During the observation period (5 weeks), bird performance and immune organ indices were recorded. Serum samples were collected for immunological evaluation and tissue samples were collected for histopathological analysis and estimation of AgNP nanosilver residue.

Treatment with AgNPs reduced the colonization of C. perfringens in the intestine and cecum, reduced the severity of clinical signs, and reduced mortality compared to the untreated infected group. Treatment with AgNPs alleviated pathological lesions in the intestines and liver, but their residues were found in the muscle.

AgNPs have a positive impact on the integrity of gut health while having no impact on immune organs. AgNPs have some residue in muscle; Therefore, further studies on the concentration and size of AgNPs, route of administration, and discontinuation time are needed to ensure the safety of chicken meat for consumers.

The broiler industry is important in Egypt, but the country is now facing the challenge of diarrheal infections, a common condition that threatens poultry production.  1 Necrotic enteritis (NE) in chickens is caused by Clostridium perfringens , a gram-positive, spore-forming, anaerobic, rod-shaped bacterium.  2 , 3 C. perfringens is classified into five toxin types (A–E) according to its ability to produce four different main toxins (alpha, beta, epsilon, and iota).  4, 5

Recently, new toxins (Beta2, NetB and TpeL) have been discovered, requiring an expanded classification scheme.  6 Necrotic enteritis is usually caused by C. perfringens type A and occasionally by type C. 7 C. perfringens is commonly found in the intestines of healthy poultry, usually at levels below 102 –104 CFU/g intestinal content, compared with 107–109 CFU/g in diseased chickens.  8 The impact of clinical and paraclinical NE is estimated to cost the industry $6 billion per year.  9

Acute NE affects broilers between 2 and 6 weeks of age, causing 1% mortality per day, with a cumulative mortality rate of up to 10–40%.  3 , 10 Subclinical NE leads to damage to the intestinal mucosa leading to reduced digestion and absorption, reduced weight gain, and increased feed conversion ratio (FCR). It can also cause hepatitis or cholangiohepatitis.  11, 12

C.perfringens causes severe foodborne enteritis in humans, 13and its presence in human foods, such as chicken, may be inevitable.  Histopathologically, NE is characterized by severe enterocyte necrosis, widespread villous fusion, and a severe inflammatory reaction in the lamina propria with severe dilatation of blood capillaries associated with Small hemorrhages in most villi, especially in the duodenum and jejunum.

In the liver, NE causes severe obstruction, portal hepatitis associated with hepatocellular degeneration and necrosis. In the cecal tonsils, it leads to severe depletion of lymphoid tissue and lymphocytosis.  15 , 16 Antibiotics increase the resistance rate of intestinal bacteria in broiler chickens, leading to antibiotic resistance in enteric pathogens of animal origin, especially C. perfringens .  17 In a previous study, 125 strains of C. perfringens obtained from clinical broilers infected with NE exhibited complete multidrug resistance to streptomycin, gentamicin, lincomycin, erythromycin, spiramycin, and oxolinic acid and partial resistance to spectinomycin, tylosin-fosfomycin, ciprofloxacin, rifampicin, chloramphenicol, enrofloxacin, neomycin, colistin, pefloxacin, doxycycline, norfloxacin, oxytetracycline, flumequine, and trimethoprim–sulfamethoxazole.  17

Nanotechnology is now providing new tools to help improve animal health and productivity as well as overcome the problem of multidrug resistance.  18 Silver nanoparticles (AgNPs) have various applications in poultry production, for example, they are used in disinfectant preparations in hatcheries, 19 and they are exploited for their antibacterial effects against many gram-negative and gram-positive bacteria;  19–24 furthermore, they exhibit antibacterial activity against Campylobacter jejuni, Escherichia coli ( E. coli), Bacillus spp., Klebsiella pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa .  25 , 26 AgNPs have also been used as growth promoters in drinking water for broiler chickens27 and have been reported to improve their health and performance.  28 This study aimed to evaluate the antibacterial effect of AgNPs on experimentally generated NE in broiler chickens and to measure the performance and immune response of the birds.

AgNPs were prepared by chemical reduction method. Silver nitrate is reduced with sodium borohydride and sodium citrate in the presence of polyvinylpyrrolidone as a capping agent.  18

The synthesized NPs were characterized by studying the absorption spectrum of silver Nanoparticles in solution via UV-Vis spectroscopy (NanoDrop 2000) and by imaging their morphological features via electron microscopy. transmittance (TEM) (FEI Tecnai G20, Netherlands) at an accelerating voltage of 200 kV.

The pathogenic strain of C. perfringens type A used in this study was obtained from the strain bank at the Department of Poultry, Faculty of Veterinary Medicine, Cairo University. Under sterile conditions, the type A C. perfringens strain was inoculated into cooked meat medium and incubated anaerobically at 37°C for 18 hours. The resulting bacterial cells were then resuspended in phosphate-buffered saline (PBS) and colony counts were adjusted using a McFarland tube. At 14 days of age, chickens in the positive control group and chickens in the infected group treated with AgNPs were infected through the plant digestive tract with 4×10 8 CFU/mL/bird of fresh C. perfringens  prepared in PBS for 2 consecutive days in accordance with the method described by Awaad et al.  29

A total of 120 Cobb chicks (1 day old) were obtained from Cairo Poultry Company, Egypt and were raised on a deep bedding system with fresh wood chips as bedding ~10 cm thick on a concrete floor. . Chicks were reared under optimal temperature, humidity, and ventilation conditions and maintained under 24-hour continuous light throughout the observation period (5 weeks). Chickens receive a balanced diet (including starter, grower and finisher diets) without any additives and clean water ad libitum. At 14 days old, 120 chicks were randomly divided into 4 equal groups (30 chicks each). Each group was divided into 3 equal replicates (10 animals/repeat). Groups were assigned as follows: infected with positive control (G1), infected and treated with AgNPs (G2), treated with AgNPs (G3), and negative control (G4). The vaccination program for all birds includes Hitchner B1+H120 vaccine in the eye at 7 days of age, subcutaneous injection of inactivated avian influenza H5N2 vaccine at 10 days of age, and LaSota vaccine in the eye at 14 days of age. days old, inject IBDV 228E vaccine into the eyes. at 18 days of age, and finally inoculation of LaSota vaccine into drinking water at 28 days of age, according to previously published protocols.  16, 29

Each chicken in groups G2 and G3 received 1 mL of AgNP suspension (30 µg/mL) via the plant gastrointestinal tract for 5 consecutive days post-infection (PI) starting at 14 days of age, resulting in a total The dose for each animal is 150 µg AgNP.

All experimental procedures and bird handling were approved from the animal care and use committee of the Faculty of Veterinary Medicine, Cairo University.

Evaluate bird performance

Morbidity and mortality rates

General health status, clinical signs and mortality were recorded daily.

Body weight and feed conversion ratio (FCR)

Regarding body weight, 15 animals/group (5 animals/replicate) were randomly selected and weighed individually because animals were weighed on weeks 2, 3, 4, 5. Feed consumption was measured on the same day chicken scale. FCR was determined according to the formula (g feed/g live body weight gain) according to Timmerman et al.  30

Sampling to enumerate C. perfringens

Three chickens were randomly selected and slaughtered according to regulations from each group (one chicken/bird) at 21, 28, and 35 days post-infection (PI).

C. perfringens counts in intestines and stools; At 7 days PI, 0.2 g from intestinal contents and feces were collected separately from each chicken (3 birds/group) (one bird/replicate) and serially diluted in PBS sterile to 1:100, 1:1000 and 1:10,000; Then, 0.1 mL of each dilution was poured onto the surface of sheep blood agar plates and tryptose sulfite cycloserine (TSC) agar (supplemented with D-cycloserine) with egg yolk emulsion and incubated anaerobically in a flask. GasPak anaerobic and GasPak 24-hour anaerobic kit. at 37°C. Typical C. perfringens colonies (black colonies) on TSC agar, or large dome-shaped colonies with double hemolytic zones on blood agar plates, were counted and reported as CFU/g. Colonies were selected and confirmed using the criteria of Harmon 31 and Garrido et al.  32

Macroscopic damage score

Macroscopic lesions were scored according to the six-point system of Keyburn et al 33 and Shojadoost et al.  34

Intestines, liver, kidneys and immune organs (thymus, bursa of Fabricius (BF) and spleen) were collected and fixed in 10% buffered formalin, then processed, sectioned and HE stained according to the following methods: described by Bancroft and Gamble.  35

Immune organ index (BF, thymus and spleen)

Immune organs are collected and weighed to calculate the index (%) as follows:

Immune organ index = immune organ weight (g) × 100/living body weight (g).

To evaluate the role of AgNPs in poultry immunity, the antibody titer of Anti-ND vaccine was estimated by collecting blood samples at 0 and 7 days after intravenous ND vaccination. The wings of 5 animals/group were randomly selected and the serum was tested. Hemagglutination inhibition (HI) test.  36

AgNP residues in muscle were determined via inductively coupled plasma optical emission spectrometry (ICP-OES).  21 In brief, muscle tissue samples (5 g) from both AgNP-treated groups (G2 and G3) were sampled at the end of the experiment (day 35). Samples were microwave digested under heat and pressure (temperature 200°C; pressure 40 bar) with the addition of 2 mL nitric acid (30%) before analysis via ICP-OES (Thermo Fisher Scientific , Older brother).

One-way analysis of variance was used to compare the effects of different treatments on growth performance parameters, immune organ index, and intestinal clostridial counts of broiler chickens. The Kruskal–Wallis test was used when data were not normally distributed (lesion scores and HI titers). Data are expressed as means and standard errors. Fisher’s exact test was used to evaluate the relationship between mortality and treatment groups. Statistical significance was set to P <0.05. PASW Statistic Software Version 18.0 (SPSS, Chicago, IL, USA) was used to conduct statistical analysis. R 3.6.1 software ( http://www.r-project.org/ ) was used to create the graph graphics.

TEM images show spherical, dispersed silver nanoparticles with an average diameter of 15 nm (Figure 1A). The UV-Vis absorption spectrum showed the maximum absorbance of the prepared AgNPs at 398 nm (Figure 1B).

Figure 1

Characteristics of silver nanoparticles. ( A ) Transmission electron microscope image shows spherical silver nanoparticles with an average diameter of 15 nm (scale bar 100 nm). ( B ) UV-vis absorption spectrum shows maximum absorption peak at 398 nm.

After experimental infection, clinical signs were clearly observed after 1 week PI in G1 chickens, manifested as generalized depression, ruffled feathers, and dark orange stools in 40% of infected chickens. In group G2, clinical signs were observed in ~15% of the chickens, while the other 2 groups showed no clinical signs.

Mortality rates in groups G1 and G2 after 1 week PI were 20% (6/30) and 3.3% (1/30), respectively, while no deaths were recorded in the remaining two groups (Table first). Compared with the positive control group (G1), mortality in the other groups decreased significantly (P = 0.004).

Note: a,b,c Mean values or percentages with different superscripts in the same column are statistically significantly different ( P < 0.05).  d Treatment: G1, chicks infected with Clostridium perfringens but not treated with nano silver; G2, chicks infected with C. perfringens and treated with nano silver; G3, uninfected chicks treated with AgNPs; G4, chicks were neither infected nor treated with AgNPs.

Abbreviations: FCR, feed conversion ratio (g feed/g gain); SEM, standard error of the mean; NS, not significant.

Performance parameters for growth are presented in Table 1. At 28 days of age, chickens treated with AgNPs (G2 and G3) had significantly higher body weight (P = 0.002) and increased (P < 0.0001) compared to control chickens (G1 and G4). Significant improvements were observed in the FCR of chickens receiving AgNPs (G2 and G3) on days 14 to 28 ( P < 0.0001).

The numbers of C. perfringen in the stool and intestine s are illustrated in Figure 2. After 7 days PI, G2, G3, and G4 showed a significant decrease in the number of C. perfringens in the cecum (P = 0.001) compared to G1. Furthermore, both G2 and G3 showed significantly lower numbers of C. perfringens in the intestine compared to G1 and G4 ( P < 0.0001).

Figure 2

Effect of nano silver on the amount of broiler feces and C. perfringens in broiler intestines (log10 CFU/g). G1: Control positive infection group; G2: Group treated with infected NPs and Ag; G3: Nano silver treated group; G4: Control negative group.  a,b Different superscripts indicate significant differences (Tukey test; P < 0.05).

Body index of immune organs (BF, thymus and spleen)

The ratio of immune organ weight/body weight is presented in table 2. There was no difference in BF index in all treatments. At 21 days of age, G2 had the lowest thymus weight ( P = 0.016), while G1 had the lowest spleen weight ( P < 0.0001). At 28 days of age, the highest spleen weight was recorded in G3 and the lowest in G1 ( P < 0.0001). At day 35, there were no significant differences between the different groups in immune organ indexes.

Table 2 Effects of AgNPs on immune organ indices (BF, thymus and spleen)

Note: a,b,c Mean values with different superscripts in the same column are statistically different ( P < 0.05).  d Treatment: G1, chicks infected with Clostridium perfringens but not treated with AgNPs; G2, chicks infected with C. perfringens and treated with AgNPs; G3, uninfected chicks treated with AgNPs; G4, chicks were neither infected nor treated with Nano silver.

Abbreviations: SEM, standard error of the mean; NS, not significant.

The HI titer values observed after ND vaccination are illustrated in Figure 3. There were no differences in HI titers between groups at 7 or 14 days PI.

Figure 3

Effect of nano silver on HI titer of broilers for NDV. G1: Control positive infection group; G2: Group treated with infected NPs and Ag; G3: AgNP treated group; G4: Control negative group. Statistical comparisons were performed using the Kruskal–Wallis Test, at a significance level of P <0.05.

Macroscopic damage score

Differences in lesion scores were observed only at day 21 (7-day PI). C. perfringens infection caused greater intestinal damage (P = 0.027) in G1 compared with G2 (fig. 4).

picture 4

Effect of Nano silver on macroscopic damage scores in broiler chickens. G1: Control positive infection group; G2: Group treated with infected NPs and Ag; G3: AgNP treated group; G4: Control negative group. a,b Different superscripts indicate significant differences (Kruskal–Wallis test; P < 0.05).

Gross intestinal damage is severe in G1; This group exhibited intestinal distension and severe intestinal obstruction from the serosal surface in some cases (Figure 5). After dissection, the opened intestine revealed the presence of foamy intestinal content mixed with undigested food particles (Figure 6). The intestinal mucosa showed varying degrees of intestinal necrosis with the presence of hemorrhagic plaques and intestinal vascular obstruction. Lesions are commonly seen in the duodenal loop and jejunum (Figure 6).

Figure 5

( A ) Group 1 small intestine shows severe intestinal obstruction. ( B ) Group 1 small intestine shows distended bowel containing gas.

Figure 6

( A ) The exposed small intestine of group 1 showed severe necrosis and intestinal mucosal stasis with large hemorrhagic plaques. ( B ) Group 1 open small intestine shows villous necrosis of the intestinal mucosa. ( C ) Open intestine of Group 1 showing the presence of undigested food particles mixed with Orange. ( D ) The open bowel of group 2 showed mild obstruction of the intestinal mucosa. ( E ) Open intestine showing normal intestinal mucosa belongs to Group 3. ( F ) Open intestine shows normal intestinal mucosa belongs to Group 4.

The liver of experimentally infected birds (G1) showed subcapsular hemorrhage, especially at the liver margin, while the intestines of birds in G2 appeared congested with an apparently normal liver (Figure 7 ). No macroscopic lesions were observed in G3 or G4.

Figure 7

( A ) Necropsy chicken carcass showing pale liver and subcapsular hemorrhage of Group 1. ( B ) Necropsy chicken carcass showing normal liver of Group 2.

Histopathological lesions were most prominent in experimentally infected birds (G1, positive control group). The epithelial layer of the intestinal villi is sloughed off, causing massive necrosis of the intestinal villi and intestinal glands. Inflammatory cells mixed with cell debris and the bacillus C. perfringens were observed (Figures 8AAnd​andB).B). In the liver, the portal vein shows severe dilation and stasis with red blood cells. The portal area showed portal edema and inflammatory cell infiltration (Figure 9A); The central vein was also obstructed (Figure 9B). There was focal concentration of mononuclear inflammatory cells in the liver parenchyma, dilatation of hepatic sinusoids and degeneration of hepatocytes. Figures 9 C and D).

Figure 8

Micrographs of chicken intestine sections: ( A and B ) Clostridium perfringens – experimentally infected group (Group 1), ( C and D ) Group infected with C. perfringens and treated with silver nanoparticles (Group 2), ( E and F ) Silver nanoparticle treated group (Group 3), ( G and H ) Negative control group (Group 4). ( A ) Massive and diffuse necrosis of intestinal villi with complete necrosis and sloughing of enterocytes (arrow) (HE, x40), ( B ) Necrotic and sloughing of enterocytes mixed with clostridial bacilli and inflammatory cells (arrow) (HE, x200), ( C ) Intestinal villi maintain integrity while the lamina propria is infiltrated by inflammatory cells (arrow) ( HE, x100), ( D ) Multiple vacuoles in enterocytes and epithelial mucosa of intestinal glands (arrows) (HE, x200), ( E ) Normal structure of intestinal villi (HE, x100), ( F ) Slight infiltration of the lamina propria with inflammatory cells (arrows) (HE, x200), ( G ) Normal histological structure of intestinal villi (HE, x100), ( H ) High magnification of the previous micrograph showing normal architecture of enterocytes and intestinal glands (HE, x200).

Figure 9

Micrographs of chicken liver: ( A–D ) Clostridium perfringens – experimentally infected group (Group 1), ( E and F ) Group infected with C. perfringens and treated with silver nanoparticles (Group 2), ( G ) Group silver nanoparticle treated (Group 3), ( H ) Negative control group (Group 4). ( A ) The portal vein is severely dilated and engorged with red blood cells and surrounded by portal edema and inflammatory cell infiltration (arrow) (HE, x100), ( B ) Central hepatic vein obstruction ( arrow) (HE, x200), ( C ) Localized concentration of mononuclear inflammatory cells in the liver parenchyma (arrow) (HE,x200), ( D ) Hepatocytes are degenerating and liver sinusoids are dilated (HE, x200), ( E ) Central vein surrounded by inflammatory cells (arrow ) (HE, x200), ( F ) Hepatic lobule and portal region showing normal pattern ( G and H ) Normal histological structure of liver lobules (HE, x200).

In the infected and nano silver-treated group, the intestine showed intact villi and intestinal glands, while the lamina propria was mildly infiltrated by inflammatory cells with numerous vacuoles in enterocytes and mucosa. intestinal gland epithelium (Figures 8C and D). The liver had normal histology with only a few central veins surrounded by inflammatory cells (Figures 9 E and F).

The AgNP-treated group exhibited normal histology of intestinal villi, enterocytes, and intestinal glands, with a small number of inflammatory cells infiltrating the lamina propria (Figures 8E ​and F), F), while the negative control group showed normal intestinal histology (Figures 8GAnd​and H).

Both the AgNP-treated group and the negative control group showed normal liver histology (Figures 9G ​and H), and the immune organs examined had no pathological lesions.

Determination of nano silver tissue residues

ICP-OES analysis at age 35 days showed the presence of residual Ag in muscle in G2 and G3 at concentrations of 413 and 501 ng/g, respectively (mean = 457 ± 44 ng/g). No residual Ag was detected in G1 or G4.

Nanotechnology has many applications in the poultry industry. In this study, characterization of nano silver through UV–Vis spectroscopy revealed a maximum absorption peak at 398 nm, corresponding to the plasmonic absorption of nano silver and confirming the successful synthesis of them. TEM also confirmed the formation of AgNPs and revealed their morphological properties related to size and shape. In this study, treated birds were administered a total dose of 150 µg of AgNPs for 5 days. At the concentrations used, AgNPs had an antibacterial effect against C. perfringens (indicated by reduced numbers) and improved the general health status of the birds tested, although no any effect on the immune parameters examined. In a parallel study by Al-Saeedi, 38 AgNPs were administered in the drinking water of broiler chickens at different concentrations (20, 30, 40, and 50 ppm) during the entire rearing period. In that study, the authors found that AgNPs at a concentration of 50 ppm provided a significantly high value ( P < 0.05) for the BF index, as well as a significant improvement ( P < 0.05) on the number of beneficial bacteria (Lactobacilli) in jejunum and reducing the number of harmful bacteria ( E. coli ).

In this study, the clinical signs observed were generalized depression, ruffled fur, and the appearance of orange frothy stools in G1 and G2. Similar findings (varying degrees of diarrhea) have been reported in chickens experimentally infected with C. perfringens .  16

Mortality rates were 20% and 3.3% in G1 and G2, respectively. Cumulative mortality rates of 10%–40% due to NE in broilers have been reported.  2 , 10 Awaad et al. 16 documented a lower mortality rate (7.41%) in chickens experimentally infected with C. perfringens , although this difference may be due to differences in bacterial strains , dosage or experimental conditions. The significant decrease in mortality in G2 ( P = 0.004) may be due to the antibacterial effect of AgNPs against C. perfringens infection.

Differences in lesion scores were observed 7 days PI. Untreated infected chickens showed an increased number of intestinal lesions (P = 0.027; figure 4) compared to AgNP-treated groups. Our results for the positive (infected) control group are consistent with those reported by Awaad et al.  16, 29

Growth performance parameters at 28 days of age showed that chickens treated with AgNPs (G2 and G3) had significantly higher body weight ( P = 0.002) and weight gain ( P < 0.0001) compared to with control chickens (G1 and G4). Significant improvements were observed in the FCR of birds receiving nano silver (G2 and G3) on days 14 and 28 ( P < 0.0001). Małaczewska 28, 39 concluded that AgNPs improved the health of poultry and promoted their growth performance.

In this study, fecal and intestinal C. perfringens amounts after 7 days PI for G2, G3, and G4 were significantly lower ( P = 0.001) than in the infected control group (G1). Furthermore, both G2 and G3 had significantly lower numbers of C. perfringens in the intestine than G1 and G4 ( P < 0.0001). These results are consistent with those of a previous study, in which AgNPs exhibited in vitro antibacterial activity against C. botulinum type A. 40 The antibacterial effect of metal NPs may be due to the breaks down and penetrates microbial cell membranes, causing damage to microbial cells. cell wall leading to leakage of substances in the cytoplasm.  41 AgNPs have been shown to exert antibacterial inhibition through close interactions with thiol groups present in key respiratory enzymes.  42

After treatment with AgNPs, there was no significant effect on the immune response of chickens and no changes were observed in BF index. After 35 days, no significant differences were observed in all immune organ indices between all treated groups. This may be due to the application of AgNPs over a period of 5 days (day 14 to day 19), the improvement in chicken performance was mainly observed during the next 14 days (from day 14 to day 28). Additionally, there were no significant differences in HI titers between groups at 7 or 14 days PI. Kulak et al. 43 found that regular application of AgNPs (2.87 and 12.25 mg/fish) improved HI (as evidenced by increased levels of immunoglobulin and interleukin 6), and they confirmed the effect of AgNPs on the immune system. The state of the birds depends on the size and concentration of AgNPs. In contrast, Ognik and colleagues found that administration of AgNPs to chickens caused disruptions in protein catabolism, as evidenced by decreased liver enzyme activity and concentrations of important protein metabolism products. (creatinine and urea) are lower. Ahmadi et al. 45 used AgNPs as a feed additive at different concentrations (4, 8, and 12 mg AgNPs per kg diet) in broiler chickens, finding that all concentrations had digestive effects. impact on performance, health and immune response.

Observations of histological changes in chicken intestine and liver between the experimentally infected control group and the AgNP-treated group showed various differences. In the intestine, the infected group showed severe necrotic changes in villi and intestinal glands with the presence of large numbers of bacteria associated with the necrotic area, according to the usual pathological picture for C .perfringens .  46 With Nano silver treatment, intestinal villi condition is significantly improved, structure is maintained and inflammatory response is reduced. Intestinal cells have some vacuoles but have not yet reached the necrotic stage. Liver lesions caused by experimental infection are prominent in G1 and include portal and central venous congestion, inflammatory cell infiltration, and degenerative changes in hepatocytes. The occurrence of liver lesions in C. perfringens infection is due to movement of the bacteria through the damaged intestine to the liver via the portal vein.  47 The treated group had a normal histological pattern of hepatocytes and hepatic vasculature with only a few inflammatory cells around the central vein. Ahmadi et al. 48 reported that different Ag levels had no significant effect on cellular changes in broiler liver tissue.

The improvement in clinical signs combined with a reduction in bacterial load and the absence of negative histopathological changes in the intestine and liver confirmed the antibacterial activity of orally administered AgNPs against C. perfringens . These results are consistent with previous reports on the antibacterial effects of AgNPs against E. coli in chickens, 49 Pseudomonas aeruginosa and Flavobacter johnsoniae in fish, 50, 51 Salmonella spp. in sheep and goats, 21 and Staphylococcus Aureus and E. coli in in vitro studies.  52 In addition, Sawosz et al. 53 found that drinking water containing 25 mg/kg AgNPs increased the lactic acid bacteria population in quail intestines and improved intestinal health.

Accumulation of AgNPs in organs affects their biological function and weight.  43 Our findings confirm that AgNP administration to chickens leaves residual Ag in muscle at 35 days of age (mean 457 ng/g), which may lead to human exposure with AgNPs through consumption of treated chicken edible parts. Our findings agree with those of Ahmadi and Rahimi;  54 They incorporated nano silver into broiler drinking water at different concentrations (4, 8, and 12 ppm) and found residues in the edible parts of broiler muscle at all concentrations. However, in one study, oral administration of six doses of approximately 1 mg/kg AgNPs (diameter, 20 nm) did not leave any residue in the hen’s muscle but only accumulated in the liver and eggs.  55 Kulak et al. 43 found that oral administration of AgNPs in broiler chickens resulted in residues in the small intestine and liver but not in the heart or breast muscle. Concentrations of up to 100 µg Ag/L in drinking water are believed to be safe for humans without causing adverse effects.  56 We plan to perform further studies to determine the required discontinuation time after oral treatment with AgNPs.

We conclude that silver nanoparticles can reduce the colonization of C. perfringens in the intestines of broiler chickens and that they have a positive impact on performance, general health and intestinal health integrity while not affects immune organs and immune responses. Nano silver has the effect of accumulating residue in chicken meat; Therefore, further studies on the concentration and size of AgNPs, route of administration, and discontinuation time are needed to ensure the safety of processed chicken for human consumption.