Nano silver, nano copper oxide and nano Ag-chitosan were tested for ecoli-killing efficacy
Escherichia coli infection is considered to be one of the most economically important multi-system diseases in poultry farms. Some nanoparticles such as nano silver, nano chitosan and copper oxide nanoparticles are known to have high toxicity against certain bacteria. However, there are no data regarding their success against in vivo experimental E. coli infection in broilers.
Therefore, this study was designed to investigate the bactericidal effects of low doses of CuO-NP (5mg / kg body weight), Ag-NP (0.5mg / kg body weight) and Ch-Ag NP (0, 5mg / kg body weight) for E. coli experimentally infected in broilers. One hundred chicks were divided into 5 groups as follows: (1) control; (2) E. coli (4×108 CFU / ml) was challenged; (3) E. coli + CuO-NP; (4) E. coli + nano silver; (5) NPs of E. coli + Ch-Ag.
The untreated group recorded the lowest weight gain as well as the highest number of bacteria and lesion scores of all organs examined. The highest liver silver content was observed in the nano-silver treated group compared with the Ch-Ag treated group. Our results conclude that Ch-AgNPs not only have the best antibacterial effects, but also act as a growth promoter in broilers without leaving any residue in the edible organs. We recommend using Ch-AgNPs in broiler farms instead of antibiotics or inoculants.
CONCLUSION
From our results, we conclude that Ch-AgNPs have powerful bactericidal activity against E. coli bacteria, neither reducing body weight gain nor leaving a toxic residue in the muscles. and edible organs. In addition, CuO-NP not only reduces the body weight of the bird, but also causes far-reaching pathological changes in various organs associated with an increase in copper levels in those organs. Our results show that the chitosan nanoparticles are not only capable of increasing the antimicrobial effect of silver nanoparticles, but are also able to reduce their biological cohesion and toxicity in different organs.
Therefore, we strongly recommend the use of Ch-Ag NPs as an alternative antimicrobial agent to treat infections without the risk of developing resistant strains of bacteria as with antibiotics. In addition, further studies are needed to discuss the mechanism of action of chitosan nanoparticles and how it can prevent the accumulation of silver or other NPs in organs of the body. Furthermore, more studies are needed to compare the effects of metal nanoparticles and chitosan coatings to confirm the ability of Ch-NPs to detoxify or improve the efficiency of many metal nanoparticles. and metal oxides.
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INTRODUCE
The poultry industry is mainly threatened by many microorganisms, which slow down growth and cause financial misfortune. Of these, Escherichia coli was associated with various broiler manifestations and was considered a significant human foodborne pathogen [1].
E. coli contamination mainly occurs in chickens through the use of contaminated feed, cross contamination in the barn, or through slaughter and handling [2]. Therefore, more emphasis may be on minimizing E. coli and other pathogens in poultry farms to reduce pathogen contamination in processed meat [3].
To increase livestock productivity, it is important to diagnose, treat and prevent disease. Currently, vaccinations and many antibiotics are used against microorganisms, but careless use of antibiotics can pose health risks to consumers [4].
Therefore, it is interesting to study other modern safe and successful bactericidal compounds to combat bacterial infections in poultry. Recently, nanotechnology has developed as a modern promising innovation for the synthesis of nanoparticles at nanometer sizes, displaying the antibacterial effects associated with the high surface area to volume ratio of them [5].
The antibacterial properties of various metal and metal oxide nanoparticles such as copper, titanium, zinc and silver have been clearly reported [6]. Silver nanoparticles are considered to be successful antibacterial agents against certain bacteria such as E. coli, Vibrio cholera, Salmonella typhi and Pseudomonas aeruginosa [7, 8].
Copper oxide, nano-silver and nano-silver-chitosan nanosynthesis materials and methods
Isolation, identification and enumeration of bacteria strains
O78 isolate coli has previously been isolated from high mortality broilers, serotyped and detected for its virulence through the Congo red-binding assay [18]. The E. coli O78 serotypes were cultured in aerobic media at 37 ° C for 24 hours prior to use as the target organism. The dose was calculated to match 4 × 108 CFU / Ml of bacterial isolates.
Preparation of copper oxide nanoparticles
Copper oxide nanoparticles were prepared by the chemical precipitation method described by Hassanen et al., [19].
Preparation of silver nanoparticles
Silver nanoparticle colloidal solution (17 ± 5 nm) was prepared by co-precipitation process through silver nitrate reduction (AgNO3) (99.99%, Aldrich, US) using sodium borohydride (99%, Aldrich, US) under boiling conditions [20]. The concentration of silver in the prepared solution is 10mM.
Preparation of chitosan silver nanoparticles
Silver -chitosan nano (Ch-Ag NCs) was prepared by reducing silver nitrate with chitosan according to the method described by Hassanen et al., [21]. The concentration of silver in the prepared solution is 10mM.
Characterization of the nanoparticles to be modulated
Actual morphology and size of the nanoparticles were assessed by high resolution transmission electron microscopy (HR-TEM) operating at accelerated voltage of 200 kV (Tecnai G2, FEI, The Netherlands). Dynamic light scattering (DLS) technique was used to estimate the mean particle size distribution as measured by the zeta sizer (Malvern, ZS Nano, UK). The chemical structure of the prepared nanoparticles was assessed using X-ray diffraction technique (XRD). Acute toxicity study to determine LD50 of selected nanoparticles.
A total of sixty commercial chicks (Cobb 500) chicks (7 days old) were used for the determination of LD50 by Ag-NP, Ch-Ag NP and CuO-NP. They are divided into different groups according to different dosages of processed nanoparticles. The LD50 value is calculated using Weil, [22] as shown in the following equation:
Log LD50 = Log Da + d (f + 1).
Log Da = log of the lowest of the four dose levels used
d = logarithm of the geometric factor (R)
f = R- values in the table
Animal and experimental design
All procedures of the experiment were carried out according to the guidelines of the Organized Animal Care and Use Commission at Cairo University and approved by Vet-CU-IACUC (approval number: 0722019057), Cairo , Egypt.
100 mixed day chicks (Cobb 500) a day were obtained from ElHawamdya- Giza. The birds are housed in barns above straw and raised under standard sanitary conditions in a building with regulated temperature and humidity.
Birds have long-term access to potable water and receive ad libitum mixtures suitable for the feeding stage of broiler nutritional needs. The chicks were randomly divided into 5 groups of 20 each.
Group (1) was held as a control group and received a normal daily saline drink by mouth for 7 days (negative control).
Group (2) was challenged at day 7 years of age using a growing truss with 4 × 108 CFU / ml / sub of E. coli O78 serogroup in PBS for 2 consecutive days following the method described by Awaad et al. the, [23] and untreated;
group (3) were challenged and treated with 5 mg / kg body weight of CuO-NP daily by mouth for 7 days;
group (4) was challenged and treated with 0.5 mg / kg body weight of nano silver per day orally for 7 days;
group (5) were challenged and treated with 0.5 mg / kg body weight of Ch-AgNP daily by mouth for 7 days.
The dosages of the nanoparticles differed from the acute toxicity study that represented 1/20 LD50. All birds were monitored and weighed weekly during the experiment.
Sampling At 21 days of age, all birds were brought into the PM room for blood and organ sampling. Blood samples were collected aseptically from the wing veins and used freshly to count the bacteria. All birds were slaughtered by detox without the use of anesthetic to remove Fabricius liver, spleen, intestines, kidneys, heart and bursa. Some of these were stored at -80 until used for bacterial isolysis, while others were stored in 10% neutral buffered formalin for histopathological examination.
Isolation of bacteria
The homogenous blood and organs (liver and spleen) were then diluted 10 times before pouring on EMB agar for plate counting. The biochemical determination was performed using the API 20-Etest kit (bioMérieux Inc., Marcy l’Etoile, France) according to the manufacturer’s instructions [24]. Serum determination of E. coli isolates was performed by antisera of E. coli (Denka Seiken, Japan) according to the method described by Blanco et al. [25].
Examination of histopathology Formin fixed tissue samples were processed according to the conventional method and cut to 4mm to obtain paraffin-stained paraffin-stained tissue sections with H&E for examination under light microscopy for diseased tissue examination. study [26].
Microscopic grading and scoring was performed to record the extent of damage to organs under examination in different treated groups. The classification criteria for degenerative, necrotic and inflammatory lesions evaluated as absent, mild, mild, moderate and severe are as follows; 0 (normal histology), 1 (<25%), 2 (25: 50%), 3 (50: 75%), 4 (> 75% tissue lesions) according to the method described by Hassanen et al. the, [27]. While the classification scheme for multifocal lesions evaluated according to the method noted by Hassanen et al. [28] was as follows: (0) nonspecific; (1) <3 foci; (2) 3-6 foci; (3) 7-12 foci; (4)> 12 focus.
Nanoparticle content in different organs
Flame atomic absorption spectroscopy (ZEISS, AAS5, and Germany) were used to measure copper and silver content in muscle and some edible organs such as the heart, liver and spleen homogenous tissue [19]. Summary; Concentrated nitric acid and 30% H2O2 was added to 0.5 g of tissue sample and kept overnight, then heated in a microwave decomposition system (ETHOS One; Milestone, Sorisole, Italy) until complete digestion. and no color. The samples are then allowed to cool, and the remaining solutions are diluted with 2% nitric acid.
Statistical analysis
Statistical analysis was performed using SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA). Values are expressed as the mean ± SEM. Vehicle comparison between several groups was performed by one-way variance analysis (ANOVA) and an independent t-test was used to compare between the two groups. Values are considered statistically significant at P ≤ 0.05.
Results comparing copper oxide, nano silver and nano silver-chitosan
Properties of nanoparticles
HR-TEM images showed spherical CuO-NPs with an average size of about 28.9–45.6 nm (Fig. 1A). Ag-NPs showed good homogeneous spheres with an average size of 17 ± 5nm
(Figure 1B). The Ch-Ag NCs showed that spherical nanoparticles with an average particle size of 17.5 nm were uniformly distributed in the chitosan matrix.
(Figure 1C). The particle size distribution curves obtained from DLS measurements are 37,3, 17,3, and 20 nm respectively for the CuO-NP, Ag-NP and Ch-Ag NCs.
(Figures 1D, 1E, 1F). The XRD pattern of CuO-NP shows Peaks at 2θ = 32.48 °, 35.54 °, 38.64 °, 48.85 °, 61.52 °, 65.66, 66.34 and 68.02 ° are assigned to (110), (−111), (111), (- 202), (−113), (022), (−311) and (220) of CuO nanoparticles, indicating that the synthetic Cu nanoparticles have hexagonal wurtzite structure (Zincite, JCPDS 04-005-4712)
(Fig. 1G). The XRD pattern of Ag-NPs showed strong and narrow peaks at angles 38.14 °, 44.41 °, 64.61 ° and 77.74 ° 2θ corresponding to hkl parameters of (111). , (200), (220), and (311), respectively (Figure 1H).
The obtained diffraction images were compared with the standard ICCD library installed in the PDF4 software, card number: (04-003-5625). The XRD sample of the Ch-Ag NCs showed that the peak of chitosan was at 2θ value of the peak about 15–35 ° wide. The silver vertices are indexed according to the face-centered cubic structure, which fits well with the JCPDS card number 04-004-8730.
The three obtained silver peaks belong to the reflections (111), (220) and (311) respectively. The results showed that the nanoparticles synthesized were silver nanoparticles because the position and relative intensity of all diffraction peaks of the samples were consistent with the crystal form of silver. The presence of chitosan, silver and the absence of impurities were evident from the XRD image (Fig. 1I).
Acute toxicity study of modulated nanoparticles
Mortality reported within 24 hours of ingestion of CuO-NP, Ag-NP and Ch-Ag NP in different treatment groups are recorded in Table (1). The calculated oral LD50 of CuO-NP, nano-silver and Ch-Ag NP in 7-day-old chicks (Cobb 500) was equal to 100, 10, 381 mg / Kg bwt per nanoparticle, respectively. Effects of different handling methods on poultry body weight and mortality. The results summarized in Figure (2) show that broiler average body weight decreased significantly in the group that did not receive the challenge treatment. There was a significant increase in body weight of chickens in the Ch-Ag NC-treated group compared with the control group. On the other hand, there was no significant difference in average body weight of birds in the nanoparticle treated group compared with the control group. The highest mortality was noted in the non-challenge group (40%), followed by the CuO-NP-treated group (20%). On the other hand, no mortality was observed in either the control group and those treated with nano-silver or Ch-Ag NPs.
Isolation of bacteria
The amount of E. coli decreased significantly in the groups treated with nano-silver or Ch-Ag NP (approximately 80%, 95%, respectively) in the blood and various organs (liver and spleen) compared with the group that did not treated. Approximately 50% reduction in the amount of E. coli in blood and organs was obtained from the CuO-NP-treated group compared to the untreated group (Table 2).
Nanoparticle Content in Muscle and Edible Organs There was a significant increase in copper, spleen and heart copper in the CuO-NP treated group compared with the control group (Table 3).
Meanwhile, a significant increase in the silver liver content of poultry in the nano silver treated group was reported. On the other hand, there was no significant difference in silver content in muscle, spleen and heart in the groups treated with nano-silver or Ch-Ag NP as compared to the control group (Table 4).
Examination of histopathology Microscopic images of all examined organs obtained from the negative control group showed normal histological structure. On the other hand, the E. coli experimental group showed severe to moderate pathological changes in all examined organs with significant improvements in all treated groups. The small intestine of the challenge group exhibited extensive acute enteritis with destruction of the villi (Figure 3A).
There is extensive degeneration and necrosis in the epithelium of the intestinal mucosa associated with significant proliferation of the cup-shaped cells (Figure 3B).
Fibrous secretions, necrotic and inflammatory cell debris are collected and form a pseudo-membrane covering the intestinal mucosa. The stroma and submucosa are severely infiltrated with heterotrophic and mononuclear inflammatory cells (Figure 3C).
Notable improvements were noted in all the treated groups, but the best microscopic imaging was observed in the Ch-Ag NPs treated groups. On the other hand, the CuO-NP-treated group showed degeneration and necrosis in some of the intestinal mucosa epithelium associated with mild to moderate inflammatory cell infiltration in the stroma and submucosa (Fig. 3D).
Despite the extensive hyperplasia of coke cells and mild inflammatory reactions observed in the intestinal mucosa layers of the nano-silver treated group (Figure 3E),
Ch-AgNP-treated group showed normal histological structure (Figure 3F).
Histological structure of intestinal mucosa using copper nanoparticles, nano silver and nano silver-chitosan
The liver of the challenged group exhibited extensive display of hepatocellular degeneration and necrosis. Multifocal clotting necrosis areas are detected and infiltrated with hepatic parenchymal replacement inflammatory cells (Figure 4A). Cholangitis is seen in some cases and is characterized by portal edema and inflammatory cell infiltrates (Figure 4B). There is a hyperplasia in the epithelium lining the bile duct related to the presence of newly formed bile ducts. Peritonitis is observed in the most part manifested by enlargement of the hepatic capsule by filamentous secretions and by inflammatory cell infiltrates. Areas with focal multifocal hemorrhage were found in the liver parenchyma (Figure 4C). The group treated with CuO-NP showed the focal area of hepatocellular necrosis infiltrated with inflammatory cells (Figure 4D). The group treated with Ag-NPs showed a moderate to diffuse cytoplasmic ability (Figure 4E). Noticeable improvements were noted in the Ch-Ag NPs-treated group and liver fractions that appeared with normal histological structures (Figure 4F).
Histological structure of the liver mucosa using copper nanoparticles, nano silver and nano silver-chitosan
The heart of the challenged group showed moderately fibrous pericarditis, characterized by blockage of blood vessels, thickening in the pericardium by fibrous secretions and inflammatory cell infiltrates (Figure 5A).
Myocardium found severe degeneration and necrosis associated with congestion and inflammatory cell infiltrates. The CuO-NP treated group showed mild to moderate degeneration and necrosis in the myocardium (Figure 5B).
On the other hand, the groups treated with nano silver (Figure 5C) or Ch-Ag NP (Figure 5D) showed significant improvements and appearance of organ with normal histological structure.
The group’s kidneys tested showed interstitial nephritis, characterized by interstitial congestion, hemorrhage, edema, and inflammatory cell infiltrates (Figure 5E).
The renal tubular epithelial cells undergo some degenerative and necrotic changes with intracellular and intracellular hyaline granules and drops. The kidneys of the CuO-NP-treated group showed moderate degeneration and necrosis of the tubular lining epithelium associated with an interstitial inflammatory response (Figure 5F).
The nano silver-treated group showed some degenerative changes in the tubular epithelium with interstitial hemorrhage (Fig. 5G). Notable improvements were observed in the Ch-Ag NP-treated group (Figure 5H) compared with the other nanoparticle-treated group, and the kidneys appeared with a normal histological structure.
Histological structure of the heart and kidney mucosa using copper, silver and silver-chitosan nano
The spleen of the challenge group showed mild to moderate pathological changes manifested by lymphocyte depletion in some lymphocytes (Figure 6A).
On the other hand, the spleen tissue sections in the nanoparticle treated groups showed normal histological structures (Figure 6B-D).
Bursa of Fabricius of the challenged group showed extensive lymphocytic depletion in most lymphocytes associated with marked thickening of the intercystic septum due to edema and inflammatory cell infiltrates (Figure 6E). ).
The CuO-NP treatment group showed moderate lympholysis with a prominent follicle septum between the shell and the marrow (Figure 6F). Although moderate lympholysis occurred in some lymphocytes of the Ag-NP-treated group (Figure 6G), the cluster obtained from the Ch-Ag NP-treated group showed structure. normal histology (Figure 6H).
Histological structure of the scrotum and spleen using nano copper, nano silver and nano silver-chitosan
Lesion scores in all examined organs of the different treated groups are illustrated in Figure. (7). The highest score was found in the E. coli challenged group, while the lowest score was found in the Ch-Ag NPs treated group. A decrease in lesion score was observed in ascending order for CuO-NP and nano silver.
Bar charts represent microscopic lesions in different organs of different treated groups
Discuss nano silver, nano silver-chitosan
The expansion of strains of bacteria that are resistant to different antibiotics has encouraged researchers as well as food business managers to look for other antibiotic options.
Nanotechnology can present a viable solution to this problem, and there are many different types of nanoparticles that are often viewed as a wide-ranging antimicrobial specialist, especially metal oxides and silver nanoparticles [29 , 30]. The antibacterial capacity of NPs is not well understood in broilers; therefore, our study was designed to investigate and compare the possible antibacterial effects of CuO, Ag and Ch-Ag nanoparticles against experimental E. coli infections in broilers. .
In this study, the challenging and untreated E. coli group showed a large reduction in body weight and this was consistent with Rosa et al., Who attributed the increased weight loss to oxidative stress due to Caused E. coli [31]. These results were reflected on the pathological and bacterial re-isolation images of the different organs of this group, showing the highest lesion scores and bacterial counts in all organs examined.
Our histopathological results were consistent with Sawah et al., Who observed fibrinolytic enteritis, fibrous pericarditis and fibrous pericarditis in E. coli-infected chickens [32 ]. Another recent study reported that E.coli infection resulted in a depletion of lymphocytes in the sac and spleen [33].
This study showed an increase in bactericidal efficiency in an increasing order for the CuO-NPs, nano-silver and Ch-Ag. Although CuO-NP has a moderate antimicrobial effect on experimental E.coli infection in broilers, it is considered toxic to broilers and induces pathological changes in all organs. is checked.
Flame atomic absorption spectroscopy results observed an increase in copper content in muscles and other edible organs, indicating that the microscopic lesions observed in the CuO-NP treated group had Relation to CuO-NP itself was not due to E. coli infection.
Indeed, CuO-NP reduced the number of bacteria in the liver, spleen and heart by about 50% compared with the untreated group. Our findings were to some extent similar to that of Jassani and Raheem, who found that CuO-NP had a significantly strong inhibitory and antimicrobial effect on E. coli [34].
Several studies have investigated the in vitro antibacterial effect of CuO-NP on food-borne pathogens such as Staphylococcus aureus, E. coli and pneumonia Klebsiella [35].
The antimicrobial activity of CuO-NP can be attributed to a sudden decline in the integrity of bacterial cell membranes along with the release of reactive oxygen species (ROS), which contribute to the decline of a number of biomolecules. Study affects the normal cell viability [36, 37].
Instead of the CuO-NP group, the other NP-treated groups showed a marked improvement in body weight, and the best results were observed in the group treated with NPs Ch-Ag. Our findings may be related to the biological effects of both silver and chitosan on harmful gut bacteria, leading to improved growth with increased nutrient absorption [38].
In addition, NP can increase intestinal absorption and utilize essential minerals to improve growth performance by increasing surface area [39]. Our histopathological results showed an increase in the height of the villi and the depth of the fluff layer in both the silver and Ch-Ag nano-treated groups, indicating mineral and nutrient uptake. maintenance was improved in such groups. Similar to antibiotics, Ag-NPs are based on improving animal health.
That is, Ag-NP allows them a chance to consume less of the supplement during the metabolic exertion needed to control immunity and use the supplement for physiological and other beneficial purposes [ 40].
The best antimicrobial effect was seen in the Ch-Ag NP-treated group compared with the other NP-treated groups, manifested by a marked decrease in both bacterial count and lesion scores at all agency is examined. This is thought to be due to the strong antibacterial properties of both silver and chitosan nanoparticles [41, 42].
The bactericidal effect of Ag-NP can be attributed to the short-term surface charge balance of the bacterial membrane, which promotes bacterial death [43-45]. Furthermore, the generation of ROS inhibits antioxidant defense mechanisms, resulting in further membrane damage.
Chitosan is a non-toxic biofilm-forming agent, obtained from shellfish and showing amazing antimicrobial activity [46]. Du et al. found that the antimicrobial properties of chitosan have been greatly improved by incorporation of it with various metals [47].
The bactericidal effects of NPs depend on their specific physicochemical properties [48,49]. Instead of traditional antibiotics, the NP has a special size <100 nm.
Their unique small size results in new properties, such as more prominent interactions with cells due to their larger surface particle mass ratio and flexible and controllable application [50,51] [50,51] . The present study showed a significant increase in the liver silver content of birds in the nano-silver-treated group compared to the Ch-Ag NP-treated birds.
Our findings suggest that the accumulation of nano-silver in broiler liver can be passed on to the consumer leading to a number of side effects. These results are consistent with some previous studies showing a significant increase in the amount of Ag stored in the liver than in muscle tissue and other organs in broilers [52, 53].
The chitosan nanoparticles contain unique functional groups (amine groups) that interact with silver ions, in addition, the nanoparticles also act as capping sites to stabilize the nanoparticles [54]. Furthermore, Ch-NP not only acts as a substrate or capping agent, but also acts as a stabilizer for Ag-NP by forming a network on the NP surface allowing a uniform silver nanoparticle distribution on the surface. face, no visible agglutination effect [55].
This, in turn, determines the potential bactericidal effect of the NP-Ch-Ags and their bioavailability by coating the outer surface of the carrier [56]. We propose that the silvering of the NP core with chitosan can reduce the agglomeration of particles and improve their solubility, bioavailability and stability, thus preventing the accumulation of silver. in organs and increase their secretion.
Conclusion
From our results, we conclude that Ch-Ag NPs have strong bactericidal activity against E. coli bacteria, neither reducing body weight gain nor leaving toxic residue in muscles and organs are edible. In addition, CuO-NP not only reduces the body weight of the bird, but also causes far-reaching pathological changes in various organs associated with an increase in copper levels in those organs. Our results show that the chitosan nanoparticles are not only capable of increasing the antibacterial effects of Ag-NP, but are also able to reduce their biological cohesion and toxicity in different organs.
Therefore, we strongly recommend the use of Ch-Ag NPs as an alternative antimicrobial agent to treat infections without the risk of developing resistant strains of bacteria as with antibiotics. In addition, further studies are needed to discuss the mechanism of action of chitosan nanoparticles and how it can prevent the accumulation of silver or other NPs in organs of the body. Furthermore, more studies are needed to compare the effects of metal nanoparticles and chitosan coatings to confirm the ability of Ch-NPs to detoxify or improve the efficiency of many metal nanoparticles. and metal oxides.
Eman I. Hassanen1 ⃰, Eman A. Morsy2, Ahmed M. Hussien3, Khaled Y. Farroh4,
Merhan E. Ali1