Antibacterial Effect of Zinc Oxide and Copper Oxide Nanoparticles as Substitute of Antibiotics against Fowl Typhoid in Broilers

this research work


INTRODUCTION
P oultry has become a dynamic livestock subsector that plays a crucial role in the economies of developing countries. It fulfills not only daily protein requirements of increasing human population in terms of high-quality food items like chicken meat and eggs but also triggers generation of employment sources. Globally, it has become a major source of food supplies around the world (Hussain et al., 2015). However, despite its rapid growth, the poultry industry faces numerous problems and bird mortality in particular is one of the main issues affecting the sustainability of poultry production worldwide. The mortality of birds might be due to spread of infectious diseases (Ahmed et al., 2022). These infectious diseases have become a huge risk to the poultry industry in terms of drug costs and consequently bird morbidity and mortality, resulting in high economic losses for any country (Abbas et al., 2015).
Fowl typhoid caused by a bacterium, Salmonella O n l i n e

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gallinarum, is one of the pathogenic infectious diseases. The infectious diseases including fowl typhoid pose heavy economic losses to the poultry industry (Yasmin et al., 2019). Incubation period of this disease is about 4-6 days and occur in all types of birds with no exception of chicken of all ages. The birds suffering from fowl typhoid show depression, anorexia, dyspnea, weakness, droopy wings, ruffled feathers, huddling, adherence of droppings to the vent and diarrhea (Brenner et al., 2000). The birds shed the bacteria in droppings that cause the contamination of food and water (Nair et al., 2015). The disease can transmit both horizontally and vertically. The young chicks die within 5-10 days of hatching and mortality can reach up to 80% (Bhatti et al., 2013). Antibiotics like Florfenicol, Enrofloxacin, Penicillin, Erythromycin, Oxytetracycline etc. are widely used against Salmonella species infection at poultry farms which leads to the induction of antimicrobial resistance (Oloso et al., 2019). The antimicrobial resistance (AMR) is the most important consequence of antimicrobial drugs used globally against Salmonella infection in animals. The antibiotics are used as metaphylactic and prophylactic treatment in the food producing animals. These agents are also used as growth promoters in broilers and other food animals. The AMR in food producing animals is of great concern (Threlfall, 2002). The irrational use of antimicrobial agents against non-typhoid Salmonella species is leading to the induction of antimicrobial resistance in microorganism. This evolutionary process makes the virulent strains able to survive in the unfavorable drug environment (Su et al., 2004). It has been foreseen that by 2050, antibiotic-resistant pathogens may cause about 10 million deaths worldwide (Castro-Vargas et al., 2020). The salmonella species can be transferred to the human being by handling or slaughtering the infected and morbid birds (Tizard, 2004;Mouttotou et al., 2017). The discovery of alternative, preventive and treatment methods could address the problem of antimicrobial resistance (AMR), as the WHO has issued a list of bacteria that have produced AMR (Oloso et al., 2019) and in the global plan of action 2015 instructions to overcome the problem and proposed to develop new drugs as antimicrobial solutions.
Nanotechnology could be a viable alternative solution for destroying fowl typhoid bacteria. The CuO and ZnO nanoparticles have sufficient bactericidal activity against a variety of gram-positive and gram-negative bacteria (Zarrindokht and Pegah, 2011;Das et al., 2013;Khashan et al., 2016) and these metal oxides ions like Zn 2+ and Cu 2+ which react with the negatively charged bacterial cells. Reactive oxygen species are produced by the nanoparticles, which bind to the bacterial cell wall, enter the cell and consequently destroy the bacterial cell (Ahmed et al., 2022). The nanoparticles (NPs) can also cause bacterial cell death by destroying the vital enzymes in the bacterial cells (Dadi et al., 2019) and after entering into the bacterial cell, ZnO NPs interact with the sulphur and phosphorus containing compounds like DNA of the bacterial cell leading to bacterial cell death (Raguvaran et al., 2015).
Keeping in view the importance of ZnO and CuO nanoparticles as an adequate alternative technique, the present study was designed with main aim to find out the optimum alternative treatment solutions replacing antibiotic. The specific objective of present study was to examine the antibacterial effect of different levels of ZnO and CuO nanoparticles in comparison with Florfenicol against Salmonella gallinarum induced infection in broilers by immunological, serum biochemistry and lipid profile parameters.

Treatment groups
One day-old broiler chicks (n= 90) were taken from a local commercial hatchery. All the experimental birds were homogenous with no visible variation regarding age, weight and size. According to ethical standards, experimental birds were given freedom from hunger and thirst as well as provided an environment in which birds expressed normal behavior. Feeding was provided uniformly to all animals as per their requirements under uniform housing and management conditions. On day 10, the birds were randomly divided into six groups; control negative, control positive, treatment 1, treatment 2, treatment 3 and treatment 4 (CN, CP, T 1 , T 2 , T 3 and T 4 ).

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Each treatment groups comprised 15 birds and kept under different individual compartments. The vaccination of the birds was administered against ND and IB on 3 rd and 14 th days of age.

Induction of infection
On day 19, the challenge of Salmonella gallinarum was given to the birds of all groups except control negative (CN) group at dose 10 8 CFU/ml via crop route method. The group was T 1 was given treatment florfenicol and groups T 2 , T 3 and T 4 were given treatments of ZnO and CuO nanoparticles at different dose level 25+10, 37.5+15 and 50+20 mg/kg/d respectively as shown in the experimental layout ( Table I). The treatment was given to the birds after the appearance of clinical signs (3 days post infection).

Parameters studied and data collection
Mortality ratio of the birds were noted between 19 th and 30 th day, whereas three birds from each group were slaughtered for sample collection on 26 th and 30 th day of trial. In order to separate the serum, the blood samples were collected in 5 ml syringes and kept in a hot air oven (37° C) for 30 min. In a 1.5 ml Eppendorf tube, the serum was collected and kept at -20° C for 15 days. The total serum proteins were determined by using Bioclin Kit, Brazil, LOT-1038 and serum albumin were determined by using Bioclin Kit, Brazil, LOT-0127. The serum globulin was determined by subtracting serum albumin from total serum proteins using following equation.

Serum globulin = Total serum proteins -Serum albuin
The lipid profile (triglycerides, high density lipids, low density lipids, very low-density lipids and total cholesterol) was determined by using commercially available kits. The triglyceride level was determined by using LabKit, Spain, LOT: LIQ-418-A, HDL-C was determined by using Human Diagnostic Kit, Germany, LOT: 0072 and total cholesterol was determined by using Human Diagnostic Kit, Germany, LOT: 0166. The VLDL were determined by dividing the triglyceride value by 5 by using following equation.
Low density lipid cholesterol was also calculated by following equation.

Antibody titer against sheep RBCs
The antibody response against sheep RBS was determined as described previously (Delhanty and Solomon, 1966). The 3% washed sheep RBCs were injected to the birds on 14 th and 21 st day of experiment. The antibody titer against sheep RBCs was determined from the serum collected from birds in all treatment groups on 21 st and 28 th day of trail. The sheep blood was collected in the EDTA vacutainer (Lab Vac, LOT: 07072014) from jugular vein of a sheep maintained at UAF Small Ruminant Farm using a sterile syring. After washing the sheep RBCs, a 3% (V/V) suspension of sheep RBCs was

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prepared in the normal saline. On day 14 th and 21 st , 1 ml of 3% sheep RBCs were injected aseptically in wing vein of birds of all groups and the serum was separated from the blood taken from the injected birds after 7-days and 14days post injection. The antibody titer against sheep RBCs was determined by testing the collected serum samples using the titration method following the inactivation of serum in hot air oven. A volume of 50 µL of phosphate buffer saline was added in well of the row of microtitration plate for each sample. A volume of 50 µL of inactivated serum sample was added in the 1 st well of the microtitration plate and incubated for 30 min at 37° C. After 30 min incubation, two-fold serial dilution was done for each sample. A volume of 50 µL of 3% sheep RBCs were added into each well and incubated at 37° C for 30 min and the titers were noted down. The treatment of collected serum with 2-merceptoethanol was done to determine the IgM and the level of IgG was determined from total antibody response minus IgM.

Statistical analysis
The collected data were analyzed under complete randomized design through ANOVA technique, whereas group mean comparison was made through Tukey's test (Steel et al., 1997) using SAS® University Edition online software SAS 15.1.

RESULTS
Effect of Florfenicol antibiotic and varying treatment levels of nanoparticles was determined in the experimental birds with induced infection of Salmonella gallinarum in terms of following studied parameters serum biochemistry, immunoglobulins and mortality percentage in Tables II, III and Figure 1, respectively.

Mortality rate
Effect of different levels of nanoparticles with comparison of Florfenicol antibiotic in terms of mortality rate is mentioned in Figure 1. Mortality was found Nil in control negative group, whereas it was at its highest i.e., 60% in control positive group. However, 20% mortality was observed in birds treated with Florfenicol, whereas 20%, 13% and 20% mortality was found in birds treated with nanoparticle treatment groups; T 2 , T 3 and T 4 , respectively. The lowest mortality was found in treatment T 3 : ZnO 37.5 + CuO 15 mg/kg/d.

Total serum proteins
At 7 th day post infection, the total serum protein was decreased (p<0.05) in control positive group as compared to control negative group. The total serum protein was higher in the group treated with antibiotic (Florfenicol) therapy. The total serum proteins were higher (p<0.05) in T 1 as compared to that of control positive group. The total serum proteins in the birds in groups treated with different levels of ZnO and CuO nanoparticles T 2 , T 3 and T 4 were found comparable (p>0.05) to that of treatment group T 1 (Florfenicol). At 11 th day post infection, the total serum protein was decreased (p<0.05) in control positive group in comparison to control negative group. The total serum proteins in the treatment groups (T 1 , T 2 , T 3 ) were found not different (p>0.05) to that of control positive group whereas total serum proteins of only treatment group T 4 were found different as compared to that of control positive group. However, the total serum proteins of nanoparticles treated groups (T 2 , T 3 T 4 ) were found comparable (p>0.05) to that of T 1 (Florfenicol) group.

Serum albumin
At 7 th day post infection, serum albumin was decreased (p<0.05) in control positive group as compared to control negative group. The serum albumin was higher through antibiotic (Florfenicol) therapy. The serum albumin was higher (p<0.05) in T 1 as compared to that of control positive group. The serum albumin of treatment groups T 2 , T 3 and T 4 was found not different (p>0.05) to that of control positive group. At 11 th day post infection, the serum albumin was decreased (p<0.05) in control positive group as compared to control negative group. The serum albumin in T 1 (Florfenicol) and T 4 was improved numerically. The serum albumin in treatment groups T 2 , T 3 and T 4 was found comparable (p>0.05) to that of T 1 .

Serum globulins
At 7 th day post infection, the serum globulins of groups T 2 and T 3 were found higher (p<0.05) as compared to that of control positive group while the serum globulins of T 1 and T 4 was found numerically higher (p>0.05) to that of control positive group. The serum globulin concentration in nanoparticle treated groups (T 2 , T 3 and T 4 ) was found comparable (p>0.05) to that of treatment group T 1 (Florfenicol). However, at 11 th day post infection, the serum globulins were found not different (p>0.05) in all groups (CN, CP, T 1 , T 2 , T 3 and T 4 ).

Total glycerides
At 7 th day post infection, the total glycerides were decreased (p<0.05) in control positive group as compared to control negative group. The total glycerides were recovered through antibiotic (Florfenicol) therapy. The total glycerides were higher (p<0.05) in T 1 as compared to that of in control positive group. The total glycerides in the groups T 2 , T 3 and T 4 were found lower (p<0.05) than that of control positive group. The total glycerides of treatment groups T 2 and T 3 were found comparable to O n l i n e

M.A. Raza et al.
that of T 1 (Florfenicol). At 11 th day post infection, the total glycerides were decreased (p<0.05) in control positive group as compared to control negative group. The total glycerides were recovered through antibiotic (Florfenicol) therapy. The total glycerides were higher (p<0.05) in T 1 as compared to that of in control positive group. The total glycerides in the groups T 2 , T 3 and T 4 were found higher (p<0.05) to that of control positive group. The total glycerides of treatment groups T 2 , T 3 and T 4 were found comparable (p>0.05) to that of T 1 (Florfenicol).

Very low-density lipids
At 7 th day post infection, very low-density lipids were decreased (p<0.05) in control positive group as compared to control negative group. Very low-density lipids were improved through antibiotic (Florfenicol) therapy. Very low-density lipids were higher (p<0.05) in T 1 as compared to that of in control positive group. Very low-density lipids of nanoparticles treated (T 2 , T 3 and T 4 ) groups were found higher (p<0.05) as compared to that of control positive group. Very low-density lipids of nanoparticles treated (T 2 and T 3 ) groups were found comparable (p<0.05) to that of group T 1 (Florfenicol) whereas very low-density lipids of treatment group T 4 was found higher (p<0.05) as compared to that of T 1 (Florfenicol). At 11 th day post infection, very low-density lipids were decreased (p<0.05) in control positive group as compared to control negative group. Very low-density lipids were recovered through antibiotic (Florfenicol) therapy. Very low-density lipids were higher (p<0.05) in T 1 as compared to that of in control positive group. Very low-density lipids of nanoparticles treated (T 2 , T 3 and T 4 ) groups were found different (p<0.05) as compared to that of control positive group. Very lowdensity lipids of nanoparticles treated (T 2 , T 3 and T 4 ) groups were found comparable (p<0.05) to that of group T 1 (Florfenicol).

High density lipids
At 7 th day post infection, high density lipids were decreased (p<0.05) in control positive group as compared to control negative group. High density lipids were improved numerically in the group T 1 . High density lipids in treatment groups T 2 , T 3 and T 4 were found different (p<0.05) to that of control positive and comparable to that of T 1 (Florfenicol). At 11 th day post infection, high density lipids were decreased (p<0.05) in control positive group as compared to control negative group. High density lipids were improved numerically in the group T 1 . High density lipids in treatment groups T 2 , T 3 and T 4 were found higher (p<0.05) to that of control positive and comparable (P>0.05) to that of T 1 (Florfenicol).

Total cholesterol
At 7 th day post infection, total cholesterol was decreased (p<0.05) in control positive group as compared to control negative group. High density lipids were improved numerically in the group T 1 . The total cholesterol of treatment groups T 2 , T 3 and T 4 was found different (p<0.05) as compared to that of control positive group and comparable (p>0.05) to that of T 1 (Florfenicol) treatment group. At 11 th day post infection, total cholesterol was decreased (p<0.05) in control positive group as compared to control negative group. Total cholesterol was higher (p<0.05) in the group T 1 as a result of antibiotic (Florfenicol) therapy. The total cholesterol of treatment groups T 2 , T 3 and T 4 was found higher (p<0.05) as compared to that of control positive group and comparable (p>0.05) to that of T 1 (Florfenicol) treatment group.

Low density lipids
At 7 th day post infection, low density lipids were decreased (p<0.05) in control positive group as compared to control negative group. Low density lipids were improved numerically in the group T 1 . Low density lipids of treatment groups T 2 , T 3 and T 4 was found higher (p<0.05) as compared to that of control positive group and comparable (p>0.05) to that of T 1 (Florfenicol) treatment group. At 11 th day post infection, low density lipids of all groups were found not different (p>0.05).

Antibody titer against sheep RBCs
The log antibody titer (Ig, IgG and IgM) of all treatment groups (T 1 , T 2 , T 3 and T 4 ) including control negative and control positive against sheep RBCs after 1 st and 2 nd injection was found not different (p>0.05) as mentioned (Table III). However, IgG in birds treated with T 3 was found higher (p<0.05) than that of control positive treatment in case of 2 nd injection of washed Sheep RBCs. While in case of treatment groups T 1 , T 2 and T 4 , the IgG level was found comparable (p>0.05) to that of control positive group. The IgG level in nanoparticles treated groups (T 2 , T 3 and T 4 ) was comparable to that of treatment group T 1 (Florfenicol).

DISCUSSION
In continuation of previous efforts, findings of this study also substantially endorsed nanotechnology as a reasonable substitute of antibiotic treatment against fowl typhoid. Preliminary findings like clinical signs appeared in response to induced infection of Salmonella gallinarum like fatigue, loss of appetite, ruffled feathers, sunken eyes, yellow diarrhea and significant mortality were in line to with the previous studies (Shivaprasad, 2000;Shah et

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Nanoparticles as Substitute of Antibiotics against Fowl Typhoid al., 2013;Chiroma et al., 2017;Birhanu et al., 2020). In addition to above, gross pathological signs like bronze colored liver, splenomegaly and necrotic foci on visceral organs; liver, spleen and heart were also validated by Kumari et al. (2013). Followed by signs, significant decline in level of serum biochemistry parameters like total serum proteins, serum albumin and serum globulins was reported by previous workers (Kokosharov, 2006;Shah et al., 2013;Fotouh et al., 2014;Biazus et al., 2017). This decile might be due to damaged liver resulting in decreased synthesis of plasma proteins and serum albumin (Biazus et al., 2017), whereas damaged kidney led to increased loss of proteins and decreased appetite (Coles, 1980). In fact catalase enzyme might be produced by Salmonella gallinarum which could trigger proteolysis and consequently it might reduce protein concentration in the blood (Kokosharov, 2000). Nanoparticles might be substantiated substitute of Florfenicol antibiotic therapy due to its adequate efficacy against induced infection of Salmonella gallinarum as evident by findings of this study. Total serum proteins and globulin level was preliminary decreased in response to infection and then reinstated might be attributed to a factor of substantial response of nanoparticles under T2 and T3 treatments at 1 st sampling. This might be due to bactericidal activity of nanoparticle by degenerating the bacterial cells (Dadi et al., 2019;Ahmed et al., 2022) which could prevent liver damage in case of treated birds.
Comparable efficacy of nanoparticles with that of antibiotic in terms of lipid profile was also noticed. Following pattern of serum proteins and globulin, lipid profile parameters; total cholesterol, triglycerides, high density lipids, low density lipids and very low density lipids were decreased and then reinstated. This might be attributed due to a factor that liver tissue exposed to nanoparticle treatment could significantly increase lipid peroxides formation (Syama et al., 2013). Furthermore, Ahmadi et al. (2013) also strengthened findings of this study that lipid profile in terms of cholesterol, HDL and LDL were increased by nanoparticle treatment in birds.
The log antibody titer against sheep RBCs was found not different (P>0.05) in our study in all treatment groups at the time of both samplings (7 th and 11 th days post infection). These findings were in line to a recent study of Ahmed et al. (2022). However, Bami et al. (2018) reported differently in contrast to findings of this study that there was an increase in the log antibody titer against sheep RBCs in birds supplemented with ZnO NPs. The reason for this divergence might be due to different methodology adopted in that study in which birds were not induced infection of Salmonella gallinarum. Further research is recommended to authenticate the efficacy of nanoparticle treatment in birds infected with Salmonella gallinarum.
It was inferred based on fact of comparable efficacy of nanoparticles with that of Florfenicol against fowl typhoid, any dose level of zinc oxide and copper oxide 25+10, 37.5+15 and 50+20 mg/kg/d could be used as substitute of antibiotic treatment. However, a combination of zinc oxide and copper oxide with concentration 37.5 + 15 mg/kg/d was found optimum level of nanoparticles based on findings regarding minimum mortality of birds infected with Salmonella gallinarum in this treatment group. In general, nanoparticles could be a potential technique to be industrialized and applied in poultry industry to save birds from the danger of fowl typhoid with no antibiotic. Replacing antibiotic with more friendly and safer treatment with nanoparticles could ensure food safety for human consumption.

CONCLUSION
It was concluded on the basis of findings of current study that nanoparticles zinc oxide and copper oxide mixture (37.5 + 15 mg/Kg/d) was found optimum alternate to Florfenicol antibiotic against Salmonella gallinarum infection in broiler birds. Hence, Zinc oxide and copper oxide nanoparticles could be an adequate alternative treatment replacing antibiotics against fowl typhoid in broilers. Further research is required to authenticate findings of present study. It is also recommended that replacing antibiotic with administration of nanoparticles through water should be studied which would be more easy for farmers for implementation. is also extended to Dr. Manshaad Basheer (Big Bird) for sponsoring in purchase of experimental birds.

Funding
A partial funding from Dr. Manshaad Basheer (Big Bird) was received in term of free experimental birds to support researchers for execution of this research trail.

IRB approval
The

Ethical statement
During the whole period research trail, the bird were provided freedom from hunger, thirst and pain. A suitable environment was provided to express the natural behavior.

Statement of conflict of interests
The authors have declared no conflict of interest.

O n l i n e F i r s t A r t i c l e
Nanoparticles as Substitute of Antibiotics against Fowl Typhoid