Effects of Silver Nitrate as Alternative to Antibiotic on Production Performance, Bacterial Count and Intestinal Histological Features of Broiler under Escherichia coli Challenge

Daud Khan1, Naila Chand1, Muhammad Saeed2, Muhammad Tahir3, Alam Zeb4 and Rifat Ullah Khan5* 1Department of Poultry Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan 2Department of Poultry Science, Cholistan University of Veterinary and Animal Science, Bahawalpur, 6300, Pakistan 3Department of Animal Nutrition, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan 4Directorate General (Extension), Livestock and Dairy Development Department Khyber Pakhtunkhwa, Peshawar, Pakistan; 5College of Veterinary Sciences, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan Article Information Received 23 February 2022 Revised 05 March 2022 Accepted 31 March 2022 Available online 16 June 2022 (early access)


INTRODUCTION
O ne of the most likely causes of antibiotic resistance microorganisms is the use of antibiotics in livestock production . Antibiotics are fed to the birds, often at subtherapeutic doses, to enhance production efficiency, allowing bacterial populations to develop genetic resistance. Because of the development of antimicrobial resistance, the use of antibiotic as growth promoters has been discouraged. The prohibition of antibiotics in Europe was quickly followed by epidemic of bacterial infections. Probiotics and prebiotics Tufail et al., 2019), organic acids Abudabos et al., 2017), phytobiotics (Ali et al., 2019;Wahab et al., 2019;Ahmad et al., 2020;Alam et al., 2020;Hafeez et al., 2020;Khan et al., 2021;Ullah et al., 2022), vitamins, minerals and other compounds (Khan et al., 2014Hafeez et al., 2021) have all been studied as alternatives O n l i n e

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to antibiotics. Colibacillosis caused by E. coli is a prevalent bacterial illness that has economic implications in poultry due to decreased production, higher mortality, and the expense of prevention and treatment. E. coli is a severe public-health concern because they may be transmitted to people via the food chain or through direct contact with sick birds. Furthermore, resistant E. coli might serve as carriers for other diseases (Ibrahim et al., 2019). Management treatments, infection control, and immunization techniques are all important aspects of avian colibacillosis control (Lutful-Kabir, 2010). In the treatment of chicken colibacillosis, a variety of antibiotic drugs are employed. Antibiotic drugs are often used, resulting in selection pressure that leads to antimicrobial resistance in E. coli (Zakeri and Kashefi, 2012).
Silver (Ag + ) is a lustrous white metal and known for antimicrobial characteristics even before penicillin was discovered (Klasen, 2000). Silver ions are very efficient against germs, do little damage to the host, and rarely develop resistance. Due to its multifactorial antibacterial activities, silver is an effective tool against wide range bacteria (Hindi et al., 2009). The NRC (2005) concluded that the greatest permissible level of silver in chicken with no deleterious effects. There is little research on the potential for ionic silver to have less negative impacts on broiler or possible in vivo effectiveness in a chicken disease model. This is mostly due to the reason that ionic silver may be more harmful for the host than particulate silver, since the dissociation rate of Ag + is linked with its biological activity. Oral supplementation of silver nanoparticles has effect to silver acetate and silver nitrate (Loeschner et al., 2011;van der Zande et al., 2012) in terms of in vivo antimicrobial action (Bouwmeester et al., 2011;Williams et al., 2015).
Silver in particulate form has recently been evaluated as a supplement in poultry as an antibiotic to improve health and growth (Sawosz et al., 2007(Sawosz et al., , 2009Chauke and Siebrits, 2012;Pineda et al., 2012;Vadalasetty et al., 2018). Because silver ion has antibacterial properties, it has been postulated that it might alter the microbiota makeup of the gastrointestinal tract (GIT), boost feed consumption, and hence improve avian performance in a way similar to antibiotic growth promotion. The objective of the present study is to evaluate silver nitrate as a potential alternative to antibiotic in broiler. Therefore, the goal of this research was to find different concentrations of silver nitrate on growth performance, bacterial count, histpathological changes in dimensions of villus and gut pH in broilers experimentally challenged with E. coli.

MATERIALS AND METHODS
All procedures in this study were approved by The Committee on Ethics and Animal Welfare, The University of Agriculture, Peshawar, Pakistan.

Birds husbandry and experimental design
A total of 800 one day old chicks of male sex were procured. After one week, chicks were weighed individually and assigned to 5 experimental groups: (1) negative control (NC) group, (2) positive control group (birds were orally challenged with E. coli O 157 H 7 at the rate of 1 × 10 9 CFU/ml), (3) infected + enrofloxacin goup (challenged with E. coli O 157 H 7 + enrofloxacin (1 mg/kg), (4) infected + SN100 group (challenged with E. coli O 157 H 7 + silver nitrate 100 mg/kg and (5) infected + SN 150 group (challenged with E. coli O 157 H 7 + silver nitrate 150 mg/kg). All treatments began on day 7 of the experiment. The chickens were kept in stainless-steel wire cages with a feeder and a drinker, which provided ad libitum feed (Ali et al., 2019) and water. The room temperature was set at 35°C for the first week and subsequently dropped to 25°C at the conclusion of the trial, using a 24-h constant-lighting regimen.

Oral challenge
The E. coli O 157 H 7 strain was cultured for 24 h at 37°C in Luria Bertani broth with shaking (120 rpm). On day 7, except NC, all birds were orally challenged with 1.0 mL (1× 10 9 cfu/mL) of freshly grown E. coli O 157 H 7 using a 1-mL pipette.

Growth performance
Birds were weighed on weekly basis to determine body weight. Feed intake was measured on daily basis. The obtained data on weight gain and feed intake was used to calculate feed conversion ratio (FCR). After the removal of the visceral organs the dressed carcass was weighed as percentage of live body weight.

Sample collection
Two birds were chosen at random from each replicate, slaughtered, and sampled. For histological analysis, tissue samples from the ilium were measured under a 40X magnification (Olympus Optical Company, Shenzhen, China) using a confocal laser scanning microscope. Each broiler had at least 15 villi that were well-oriented and undamaged when they were measured. The villus height/ crypt depth ratio was determined based on the results of these observations.
Cecal Escherichia coli About 0.5 g cecal contents were mixed with 4.5

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mL sterile buffered peptone for 1 h, and then diluted ten folds in sterile buffered peptone for an additional h. E. coli was isolated from cecal contents by plating them on MacConkey's agar at 37°C for 24 h and then plating them on MRS agar under anaerobic conditions at 37°C for 24 h. Bacteria were enumerated and represented as total cfu/g digesta, and the log10-transformed data was presented.

Statistical analysis
All data were analyzed using a one-way ANOVA in a totally randomized design utilizing statistical software (Statistix" version 17). All means are reported as leastsquares means (SEM). The treatment was incorporated as a fixed effect in the statistical model at each stage of the experiment. Before analysis, the data for cecal bacterial enumeration were converted using the log 10 transformation. The Tukey test was used to test if there were statistical differences between treatments. In order to be declared statistical significant, a P-value of less than 0.05 was required.

Growth performance
On a weekly basis and over all feed intake data of Escherichia coli challenged broilers fed changed levels of silver nitrate is given in Table I. During 2 nd week of age feed intake was not significantly different (p>0.05) among the groups. In the trial of the 3 rd week, the maximum feed intake (g) data was noted in NC, Infected + enrofloxacin and Infected + SN100. Infected +SN150 and PC showed the lowest feed intake data. During 4 th , significantly (P<0.01) higher feed intake was recorded in NC followed by Infected + enrofloxacin and Infected + SN100. During 5 th week and overall, significantly (P<0.01) higher feed intake was noted in NC, Infected + enrofloxacin and Infected + SN100. Significantly (p<0.01) lower feed intake was found in PC and infected +SN150 in all stages except week 2. Overall feed intake was significantly (P<0.05) higher in infected +SN100 compared to positive control.
The effect of different levels of silver nitrate on body weight gain (g) in E. coli challenged broilers is shown in Table II. Among all groups during 2 nd week no significant difference was noted in body weight. In the 3 rd and 4 th weeks, significantly (p<0.01) higher weight gain was found NC followed by Infected + enrofloxacin and Infected + SN100. Significantly (p<0.01) higher weight gain was recorded in NC followed by infected + enrofloxacin, infected + SN100 and infected + SN150. Overall weight gain was significantly (p<0.01) higher in NC followed by infected + enrofloxacin and infected + SN100. Lowest (p<0.01) weight gain was recorded in PC and infected + SN150.   The efficacy of different levels of silver nitrate on FCR in E. coli challenged broilers is presented in Table III. During 2 nd week of age there was no significant difference in FCR among all groups. Significantly (p<0.05) better FCR was found in NC compared to PC in 3 rd , 4 th , 5 th weeks and overall basis. Overall FCR was significantly (P<0.05) higher in infected +SN100 compared to positive control.

Carcass yield and fecal E. coli count
On dressing percentage, the effect of different levels of silver nitrate in E. coli challenged broiler chicks is shown in Table IV. Dressing percentage was significantly (p<0.01) higher in NC followed by infected + SN100 and infected + enrofloxacin. The lowest dressing percentage was found in PC and infected + SN150. Similarly, E. coli in feces was also significantly (p<0.01) higher in PC and infected + SN150. E. coli count was similar in infected + SN100 and infected + SN150 (Table IV).

Histological dimensions of ilium
Effects of different levels of silver nitrate on histology of ileum in broilers chicks challenged with E. coli are shown in Table V. Villus height, crypt depth, goblet cells and epithelial thickness were significantly (p<0.01) higher in NC compared to PC. These parameters had statistically no significant difference between infected + SN100 and infected + enrofloxacin. Resultantly, villus height and crypt depth ratio was significantly (p<0.01) higher in PC compared to NC followed by infected + SN150.

pH of gizzard and small intestine
On pH of the gizzard and small intestine, the efficacy of different levels of silver nitrate in Escherichia coli challenged broilers is shown in Table VI. Gizzard pH was significantly (p<0.01) higher in PC compared to NC. However, pH of duodenum, jejunum and ileum was significantly (p<0.05) higher in NC compared to PC. pH in gizzard and small intestine was similar (p>0.05) in infected + SN100 and infected + enrofloxacin, however, it was significantly (p<0.01) different from infected + SN150.

DISCUSSION
In the present study, growth performance in terms of feed intake, weight gain, FCR and dressing percentage of birds supplemented with silver nitrate at the rate of 100 mg/ kg was significantly higher compared to the PC. Similar results were reported by Saleh and El-Magd (2018), who supplemented 100ppm silver nitrate in broiler ration for 12 days and reported significantly higher feed intake, weight gain and FCR in the supplemented group. Slight improve in growth was also reported by Yemdjie et al. (2017) in broilers, however, dose of silver nitrate was low (10 mg/ kg). Because silver ion has antibacterial properties, it has been postulated that it might alter the microbiota makeup of the gastrointestinal tract (GIT), boost nutrient utilisation, and hence improve bird performance in a way similar to antibiotic growth promotion. Little information is available on the use of silver nitrate on broiler production in a disease or without disease model.

O n l i n e F i r s t A r t i c l e
Effects of Silver Nitrate as Alternative to Antibiotic on Production Performance  Moreover, it is also clear that SN150 had negative effects on growth performance. There is little research on the potential for ionic silver to impacts broiler performance or possible in vivo effectiveness using a disease model in chicken. This is mostly due to the fact that ionic silver may be more harmful than particle silver, since the dissociation rate of silver ion (Ag + ) affects its biological activity. In terms of organ distribution and removal following oral administration, oral exposure to silver nanoparticles was observed to be very similar to oral exposure to silver nitrate (Loeschner et al., 2011;van der Zande et al., 2012).
In the current study, total E. colii count was significantly lower in SN100 compared to the positive control. Silver as a metal is not reactive with microorganisms unless it is ionised, and the bioactive form of silver is known as Ag + ion. Silver ions' antibacterial action is characterized by high efficacy, minimal toxicity, and a low rate of resistance development. Silver is active against multidrug resistant bacteria, under aerobic and anaerobic conditions. These mechanisms include damage to cell membrane, changing ion homeostasis and inhibiting respiratory enzymes, affecting antioxidant status which eventually leads to damage to lipids and proteins. The effect of silver ions with bacterial membranes has been identified as a key process by which silver ion toxicity is initiated (Randall et al., 2013). Previous investigations utilizing silver nitrate against E. coli under anaerobic and aerobic settings revealed that silver reduced bacteria (Park et al., 2009).
The gut is another important organ for water born toxicant exposure, and as a result, it is utilized as a biomarker in toxicological investigations. In the current study, the pH and histological structures of ilium were improved in SN-100 compared to the positive control. Further, high dose (SN-150) had negative impact on the ultral structures of intestines. In the study of Sawosz et al. (2007), administration of particulate silver had no negative impact on intestinal villi of Japanese quails. In another study, Ognik et al. (2016) reported that the jejunum histological structures were not different from that of normal when broilers were supplemented with lipid coated nanosilver hydrocolloid. Similar to our study, Salem et al. (2021) reported that silver nanoparticles significantly improved the intestinal villi and the inflammatory reaction was substantially reduced. Kumar et al. (2020) also reported that 50 ppm silver nanoparticles had no negative effects on the structural composition of intestines in broilers.

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D. Khan et al. From the results of the present study, it was concluded that silver nitrate at the rate of 100 mg/kg enhanced growth performance, carcass characteristics, decreased E. coli count and enhanced gut histological features in broilers infected with E. coli infection.

Ethical approval
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes and all the procedures with animals were approved by the Local Ethics Committee of Animal Experiments of the University (Protocol no. 2020-1-134).

Competing interest
There is no potential competing interest with this study.

Consent to participate and consent to publish
All the authors have equally participated in this study and agreed to publish this work in this journal.

Data availability
Data is available in the thesis.

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

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Effects of Silver Nitrate as Alternative to Antibiotic on Production Performance O n l i n e