Comparative assessment of the bactericidal effect of nanoparticles of copper oxide, silver, and chitosan-silver against Escherichia coli infection in broilers

Abstract Escherichia coli infection is considered one of the most economically important multi-systemic diseases in poultry farms. Several nanoparticles such as silver, chitosan, and copper oxide are known to be highly toxic to several microbes. However, there are no data concerning their success against in vivo experimental E. coli infection in broilers. Therefore, the present study was designed to investigate the bactericidal effect of low doses of CuO-NPs (5 mg/kg bwt), Ag-NPs (0.5 mg/kg bwt), and Ch-Ag NPs (0.5 mg/kg bwt) against E. coli experimental infection in broilers. One hundred chicks were divided into five groups as follows: (1) control; (2) E. coli (4 × 108 CFU/ml) challenged; (3) E. coli +CuO-NPs; (4) E. coli +Ag-NPs; (5) E. coli +Ch-Ag NPs. The challenged untreated group, not NPs treated groups, recorded the lowest weight gain as well as the highest bacterial count and lesion score in all examined organs. The highest liver content of silver was observed in Ag-NPs treated group compared with the Ch-Ag NPs treated group. Our results concluded that Ch-Ag NPs not only had the best antibacterial effects but also acted as a growth promoter in broilers without leaving any residues in edible organs. We recommend using Ch-Ag NPs in broiler farms instead of antibiotics or probiotics.


Introduction
Poultry industry is primarily threatened by numerous microorganisms, which diminish the growth rate and cause broadly financial misfortunes. Among these, Escherichia coli is related to different manifestations in broilers and viewed as a significant foodborne pathogen in humans [1]. E. coli infection mostly occurs in chickens by using contaminated food, cross-contamination in breeding houses, or through slaughter and handling [2]. Consequently, more accentuation is probably centered on diminishing E. coli and other pathogens on poultry farms to reduce contamination with pathogens in prepared meats [3]. To increase livestock productivity, it is important to make diagnosis, treatment, and prevention of diseases. Currently, immunization and numerous antibiotics are used to combat microorganisms, but careless usage of antibiotics may cause wellbeing dangers to consumers [4]. Thus, it is interesting to examine other modern sorts of secure and successful biocidal compounds to battle poultry bacterial infections. Recently, nanotechnology has developed a modern promising innovation for nanoparticle synthesis in the nanometer size, which displays antimicrobial impacts related to their high surface area-to-volume proportion [5]. The antibacterial potentials of different metal and metal oxide nanoparticles such as copper, titanium, zinc,

Bacterial re-isolation
The blood and organ (liver and spleen) tissue homogenates were then ten-fold serially diluted before platting on EMB agar for plate counting. Biochemical identification was performed using API 20-Etest kit (bioMérieux Inc., Marcy l'Etoile, France) according to the manufacturer's instructions [24]. Serological identification of the isolated E. coli was done using E. coli antisera (Denka Seiken, Japan) according to the method described by Blanco et al. [25].

Histopathological examinations
Formalin-fixed tissue specimens were processed via conventional methods and sliced into 4 mm sections to obtain paraffin-embedded tissue sections stained by H&E to be examined under light microscope for histopathological examination [26].

Nanoparticle content in different organs
Flame atomic absorption spectrophotometer (ZEISS, AAS5, and Germany) was used to measure the contents of copper and silver in muscle and some edible organs such as heart, liver, and spleen tissue homogenate [19]. Briefly, concentrated nitric acid and 30% H 2 O 2 were added to 0.5 g tissue samples and kept overnight, then heated in a microwave digestion system (ETHOS One; Milestone, Sorisole, Italy) till it became completely digested and colorless. Afterward, the samples were allowed to cool, and the remaining solutions were diluted with 2% nitric acid.

Statistical analysis
Statistical analysis was performed utilizing SPSS version 16.0 software (SPSS Inc., Chicago, IL, U.S.A.). Values were expressed as means + − SEM. Comparison of means between several groups was performed by one-way analysis of variance (ANOVA) and independent t test was used to compare between two groups. Values were considered statistically significant at P≤0.05.

Characterization of nanoparticles
HR-TEM images showed spherical-shaped CuO-NPs with average size ∼28.9-45.6 nm ( Figure 1A). Ag-NPs showed well uniformed spheres with average size of 17 + − 5 nm ( Figure 1B). Ch-Ag NCs showed spherical shaped nanoparticles with average particle size 17.5 nm distributed homogeneously in the Ch matrix ( Figure 1C). The particle size distribution curve obtained from DLS measurements were 37.3, 17.3, 20 nm for CuO-NPs, Ag-NPs, and Ch-Ag NCs, respectively ( Figure 1D Figure 1G). XRD pattern of Ag-NPs showed sharp intense and narrow peaks at 38.14 • , 44.41 • , 64.61 • , and 77.74 • 2θ angles those corresponding to hkl parameters of (111), (200), (220), and (311), respectively ( Figure 1H). The obtained diffraction pattern was compared with the standard ICCD library installed in PDF4 software, card no: (04-003-5625). XRD pattern of Ch-Ag NCs showed a peak of chitosan at 2θ value of the broad peak ∼15-35 • . The peaks of silver were indexed to the face-centered cubic structure which is in good agreement to the JCPDS card No. 04-004-8730. The three silver peaks obtained belong to the (111), (220), and (311) reflections, respectively. The results showed that the synthesized nanoparticles were Ag-NPs because the position and relative intensity of all the diffraction peaks of the samples were consistent with the crystalline pattern of  silver. The presence of chitosan, silver, and the absence of impurity phases were evident from the XRD image ( Figure  1I).

Acute toxicity study of the prepared nanoparticles
Mortalities recorded within 24 h after oral administration of CuO-NPs, Ag-NPs, and Ch-Ag NPs in different treatment groups were recorded in (Table 1). The calculated oral LD 50 of CuO-NPs, Ag-NPs and Ch-Ag NPs in 7-day-old chicks (Cobb 500) equals 100, 10, 381 mg/kg bwt for each nanoparticle, respectively.

The effect of different treatments on the body weight and mortality of birds
The results summarized in Figure 2 showed remarkable reduction in mean body weight of broiler chickens in challenged untreated group. There was a noticeable increasing in body weights of chickens in group treated with Ch-Ag NCs compared with control group. On the other hand, there was no significant difference in mean body weight of birds in nanoparticles treated groups compared with control group. The highest mortality rate was recorded in the challenged untreated group (40%) followed by CuO-NPs treated group (20%). On the other side, no mortality recorded in both control group and those treated with either Ag-NPs or Ch-Ag NPs.

Table 2 The effect of different treatments on the mean E. coli count (CFU/ml) in the blood and different organs after 21 days post challenge Blood
Liver Spleen Value was demonstrated as mean + − SEM. Value with different letters considered significantly different at P≤0.05 (n=7 birds/group). Capital letters indicate significance between all five groups while small letters indicate significance between the three nanoparticles treated groups.

Control Ag-NPs Ch-Ag NPs
Values were presented as mean + − SEM (n=7 birds/ group). Values with different letters considered significantly different at P≤0.05. Capital letters A and B indicate significance between all groups while small letters a, b, and c indicate significance between nanoparticles treated groups.

Bacterial re-isolation
There was a remarkable reduction in the E. coli count in groups treated either with Ag-NPs or Ch-Ag NPs (∼80, 95%, respectively) in blood and different organs (liver and spleen) compared with challenged untreated group. Approx. 50% reduction in the E. coli count observed in both blood and organs obtained from CuO-NPs treated group compared with the challenged untreated group (Table 2).

Nanoparticles contents in muscle and edible organs
There was a significant increase in copper contents in muscle, spleen, and heart in CuO-NPs treated group compared with the control group (Table 3). Whereas, a significant increase in silver contents of liver of birds in Ag-NPs treated group were reported. On the other hand, there were no significant difference in silver content in muscle, spleen, and heart in the groups treated with either Ag-NPs or Ch-Ag NPs compared with the control group (Table 4).

Histopathological examinations
Microscopic pictures in all examined organs obtained from the control negative group showed normal histological structures. On the other hand, the E. coli challenged group showed severe to moderate pathological alterations in all examined organs with remarkable improvements in all the treated groups.
Small intestine of the challenged group showed extensive acute enteritis with destruction of intestinal villi ( Figure  3A). There were extensive degenerations and necrosis in the epithelial lining intestinal mucosa associated with remarkable hyperplasia of the goblet cell ( Figure 3B). Fibrinous exudates, necrotic cell debris, and inflammatory cells were collected and forming pseudo-membrane covering the intestinal mucosa. Lamina propria and submucosa severely infiltrated with heterophils and mononuclear inflammatory cells ( Figure 3C). Remarkable improvements were recorded in all the treated groups, but the best microscopic picture observed in groups treated with Ch-Ag NPs. Otherwise, the group treated with CuO-NPs showed degeneration and necrosis in some of the epithelial lining intestinal mucosa associated with mild to moderate inflammatory cells infiltration in lamina propria and submucosa ( Figure 3D). In spite of extensive hyperplasia of goblet cells and mild inflammatory reactions observed within the intestinal mucosal layers of AgNP-treated group ( Figure 3E), Ch-Ag NPs treated group showed normal histological structures ( Figure 3F).
Liver of the challenged group showed extensive hepatocellular degeneration and necrosis. Multifocal areas of hepatocellular coagulative necrosis were detected and infiltrated with inflammatory cells replacing the hepatic parenchyma ( Figure 4A). Cholangiohepatitis noticed in some cases and characterized by portal edema and inflammatory cells infiltration ( Figure 4B). There was hyperplasia in the epithelial lining bile duct associated with the presence of newly formed bile ductules. Perihepatitis observed in most sections manifested by extensive widening of the hepatic capsule by fibrinous exudates and inflammatory cells infiltrations. Multifocal to coalescent areas of hemorrhage were noticed within the hepatic parenchyma ( Figure 4C). Group treated with CuO-NPs showed focal area of hepatocellular necrosis infiltrated with inflammatory cells ( Figure 4D). Group treated with Ag-NPs showed moderate to diffuse hepatocellular cytoplasmic vacuolization ( Figure 4E). Remarkable improvements were recorded in the group treated with Ch-Ag NPs and the liver sections appeared with normal histological structures ( Figure 4F).
Heart of the challenged group showed moderate fibrinous pericarditis characterized by congestion of blood vessels, thickening in the pericardium by fibrinous exudates and inflammatory cell infiltrations ( Figure 5A). Cardiac muscle noticed severe degeneration and necrosis associated with congestion and inflammatory cell infiltration. CuO-NPs treated group showed mild to moderate degeneration and necrosis in the cardiac muscle ( Figure 5B). On the other side, groups treated with either Ag-NPs ( Figure 5C) or Ch-Ag NPs ( Figure 5D) showed remarkable improvements and the organ appeared with normal histological structures.
Kidneys of the challenged group showed interstitial tubule-nephritis characterized by interstitial congestion, hemorrhage, edema, and inflammatory cells infiltration ( Figure 5E). Renal tubular epithelial cells suffered from several degenerative changes and necrosis with intracellular and intraluminal hyaline cast and droplets. Kidneys of the group treated with CuO-NPs showed moderate degeneration and necrosis in the epithelial lining renal tubules associated with interstitial inflammatory reactions ( Figure 5F). Ag-NPs treated group showed several degenerative changes in the tubular epithelium with interstitial hemorrhage ( Figure 5G). Remarkable improvements observed in the group treated with Ch-Ag NPs ( Figure 5H) compared with other nanoparticles-treated group and the kidneys appeared with normal histological structures.
Spleen of the challenged group showed mild to moderate pathological alterations manifested by lymphocytic cell depletion in some lymphoid follicles ( Figure 6A). On the other hand, spleen tissue sections in nanoparticle-treated groups showed normal histological structures ( Figure 6B-D).
Bursa of Fabricius of the challenged group showed extensive lymphocytic cell depletion in most lymphoid follicles associated with marked thickening in the inter follicular septa by edema and inflammatory cell infiltrations ( Figure  6E). Group treated with CuO-NPs showed moderate lymphocytolysis with prominent follicular septa between cortex and medulla ( Figure 6F). In spite of moderate lymphocytolysis occurred in some lymphoid follicles of Ag-NPs treated group ( Figure 6G), bursa obtained from Ch-Ag NPs treated group showed normal histological structures ( Figure 6H).
Lesion scoring in all examined organs of different treated groups was illustrated in (Figure 7). The highest score noticed in the E. coli challenged group, while the lowest score noticed in the group treated with Ch-Ag NPs. Reduction in lesion scoring observed in an ascending order for CuO-NPs and Ag-NPs.

Discussion
The expansion of bacterial strains showing resistance from different antimicrobials has encouraged researchers just as the food business administrators to search for other antimicrobial options. Nanotechnology may introduce a probable solution for this problem and there are different kinds of nanoparticles usually viewed as a wide-range antimicrobial specialist especially metal oxide and Ag-NPs [29,30]. The antibacterial potentials of NPs are poorly understood in broiler chickens; therefore, our study was designed to investigate and compare between the possible antibacterial effect of nanoparticles of CuO, Ag, and Ch-Ag against E. coli experimental infections in broiler chickens.
In the present study, E. coli challenged, and untreated groups showed extensive reduction in body weight and this is consistent with Rosa et al., who attributed the decreased weight gain to the oxidative stress caused by E. coli [31]. These results reflected on the pathological pictures and bacterial re-isolation in different organs of this group, which showed the highest lesion score and bacterial count in all examined organs. Our histopathological results were come in accordance with Sawah et al., who observed fibrinonecrotic enteritis, fibrinous perihepatitis, and fibrinous pericarditis in chickens infected with E. coli [32]. Another recent study reported that E. coli infection resulted in depletion of lymphocytes in bursa and spleen [33].
The present study showed an increase in the bactericidal effect in an ascending order for CuO-NPs, Ag-NPs, and Ch-Ag NPs. Although the CuO-NPs exert moderate antibacterial effects against E. coli experimental infection in broiler chickens, it is considered to be toxic to the broilers and elicit pathological alterations in all examined organs. Flame atomic absorption spectroscopic results observed an increase in the copper content in muscle and other edible organs, suggesting that the microscopic lesions observed in the CuO-NPs treated group is related to the CuO-NPs itself not to E. coli infections. Indeed, CuO-NPs reduced ∼50% of the bacterial count in the liver, spleen, and heart compared with the challenged untreated group. Our findings were some extent similar to Al-Jassani and Raheem, who found that CuO-NPs have considerable powerful inhibitory and anti-bacterial effect on E. coli [34]. Several studies have investigated the in vitro antimicrobial effects of CuO-NPs on different kinds of food-borne pathogens as Staphylococcus aureus, E. coli, and Klebsiella pneumonia [35]. The antibacterial activity of CuO-NPs could be attributed to the sudden decline in bacterial cell membrane integrity in addition to the release of ROS, which contribute to the degradation of several biomolecules that affect also on normal cell viability [36,37].
Rather than CuO-NPs treated group, other NP-treated groups showed marked improvements in body weights and the best results were observed in the group treated with Ch-Ag NPs. Our findings may be related to the biological   effects of both silver and chitosan on intestinal harmful bacteria, which resulted in improved growth as the absorption of nutrients was increased [38]. Additionally, NPs could increase intestinal absorption and utilization of minerals required for improving growth performance by increasing the surface area [39]. Our histopathological results showed an increase in the height of the intestinal villi and crypt depth in both Ag and Ch-Ag NPs treated groups, suggesting improved mineral and nutrient absorption in such groups. Similarly, as antibiotics, Ag-NPs are relied upon improving the well-being of animals. That is, Ag-NPs allowing them the chance to consume fewer supplements on the metabolic exertion required for immunological control and to use additional supplements for other physiological and gainful purposes [40].
The best antibacterial effect was noticed in Ch-Ag NPs treated group compared with other NPs treated groups, which manifested by a marked reduction in both bacterial count and lesion score in all examined organs. This is attributed to the potent antibacterial capacity of both silver and chitosan nanoparticles [41,42]. The bactericidal action of Ag-NPs might be because of a brief balance of the surface electric charge of the bacterial membrane promoting bacterial death [43][44][45]. Moreover, the generation of ROS restrains the antioxidant defense mechanism leading to further damage to the cell membrane. Chitosan is a nonpoisonous biopolymer obtained from shellfish and showed an amazing antibacterial activity [46]. Du et al. found that the antimicrobial properties of chitosan were improved extensively by loading it with different metals [47]. The bactericidal effects of NPs depend on their particular physicochemical properties [48,49]. Rather than traditional antibiotics, NPs have peculiar dimensions <100 nm. Their uniquely small size outcomes in novel properties, as more prominent interaction with cells because of a larger surface area-to-mass ratio and flexible and controllable application [50,51].
The current study revealed a significant increase in silver content of the liver of birds in the group treated with Ag-NPs in contrast with those treated with Ch-Ag NPs. Our findings suggest that the accumulation of Ag-NPs in the liver of broiler chickens may be transferred to consumers leading to several side effects. These results are in harmony with several previous studies showed a marked increase in Ag retention in the liver more than in muscular tissue and other organs in broiler chickens [52,53]. Chitosan nanoparticles contain unique functional groups (amino groups) that interact with silver ions, in addition, nanoparticles as well act as capping sites for nanoparticle stabilization [54]. Moreover, Ch-NPs not only act as matrix or capping agent but also act as stabilizing agent for Ag-NPs by forming a network on NP surface allows the homogeneous Ag-NPs distribution on the surface, with no visible aggregation effects [55]. This, in turn, determines the potential bactericidal effects of Ch-Ag NPs and their bioavailability by covering the external surface of the carrier [56]. We suggest that coating of silver NP core by chitosan can reduce particle aggregation and improve their solubility, bioavailability, and stability, so that, it prevents accumulation of silver in organs and increasing their excretion.

Conclusions
From our results, we concluded that Ch-Ag NPs had a powerful bactericidal activity against E. coli, which neither reduced body weight gains nor leaving toxic residues in muscles and edible organs. Alternatively, CuO-NPs not only reduced body weights of birds but also caused extensive pathological alterations in different organs associated with increasing copper levels in such organs. Our results find that chitosan nanoparticles not only have the ability to increase the antibacterial effect of Ag-NPs but also it can reduce their bioaggregation and toxicity in different organs. Therefore, we highly recommended using Ch-Ag NPs as an alternative antibacterial agent in treating infections without taking the risk of developing resistant bacterial strains as with antibiotics. In addition, further studies are required to discuss the mechanism of action of chitosan nanoparticles and how it can prevent the accumulation of silver or other NPs in body organs. Moreover, more studies are needed to compare the effect of metal nanoparticles and those coating with chitosan to confirm the ability of Ch-NPs either on detoxifying or improve the efficacy of many metallic and metal oxide nanoparticles.

Data Availability
All data will be available on request to the corresponding author.