A Comparative Study of the Effect of Non-Antibiotic Feed Additives on Experimental Colonization of Salmonella Enterica Serovar Enteridis and Intestinal Pathomorphology in Broiler Chickens

ABSTRACT The objective of this study was to evaluate the effect of eubiotics on the intestinal morphology of broilers. For this purpose, 125 birds were divided into six groups with two replicates each (10 birds in each replicate). Group A was given a Basal diet. All groups except group A were challenged with Salmonella enterica serovar Enteritidis. Group B was provided the basal diet, group C was fed a Probiotic-added diet; group D was fed a Prebiotics-based diet; group E was given essential oils plus the basal diet; and group F was provided with organic acids plus the basal diet. Two separate experiments were carried out for Salmonella recovery, checking the cecal tonsils and conducting an intestinal pathomorphic evaluation. Villus length, villus width, villus surface area, and crypt depth were measured by micrometry. There was an overall improvement (p<0.05) in intestinal morphometric parameters for all the treatment groups except for the negative control group, which showed the lowest villus height and villus depth values. Maximum villus height (p<0.05) of the duodenum was achieved by group E, which was fed a diet containing essential oils, whereas a maximum villus surface area index (p<0.05) was recorded for the birds of Group D, which were fed a diet containing prebiotics. Maximum villus height (p<0.05) and surface area index in ileum mucosa was recorded (p<0.05) in the birds of group D (treated with prebiotics). It is concluded that there is an overall increase in the gut histology of broilers fed non-antibiotic based feed.


ABSTRACT
The objective of this study was to evaluate the effect of eubiotics on the intestinal morphology of broilers.For this purpose, 125 birds were divided into six groups with two replicates each (10 birds in each replicate).Group A was given a Basal diet.All groups except group A were challenged with Salmonella enterica serovar Enteritidis.Group B was provided the basal diet, group C was fed a Probiotic-added diet; group D was fed a Prebiotics-based diet; group E was given essential oils plus the basal diet; and group F was provided with organic acids plus the basal diet.Two separate experiments were carried out for Salmonella recovery, checking the cecal tonsils and conducting an intestinal pathomorphic evaluation.Villus length, villus width, villus surface area, and crypt depth were measured by micrometry.There was an overall improvement (p<0.05) in intestinal morphometric parameters for all the treatment groups except for the negative control group, which showed the lowest villus height and villus depth values.Maximum villus height (p<0.05) of the duodenum was achieved by group E, which was fed a diet containing essential oils, whereas a maximum villus surface area index (p<0.05)was recorded for the birds of Group D, which were fed a diet containing prebiotics.Maximum villus height (p<0.05) and surface area index in ileum mucosa was recorded (p<0.05) in the birds of group D (treated with prebiotics).It is concluded that there is an overall increase in the gut histology of broilers fed non-antibiotic based feed.

INTRODUCTION:
The use of antimicrobial agents as a preventive measure has been questioned, given the extensive documentation of the evolution of antimicrobial resistance among pathogenic bacteria.Non-antibiotic feed additives (probiotics, prebiotics, essential oils, and organic acids) are being considered to fill this gap and a few farmers in the country are already using them with good results (Abbas et al., 2018;Mustafa et al., 2021a).On the other hand, the practicality of antimicrobial mediators as a prophylactic measure has been strongly questioned due to the risk of selection and emergence of antimicrobial resistance amid pathogenic microorganisms (Batool et al., 2020).Therefore, the use of antibiotics as growth promoters for poultry and the fear of residual impacts of their consumption as therapeutic mediators have created an atmosphere of debate and consumer reluctance, causing a search for alternatives (Abbas et al., 2018).The use of natural products such as plant extracts, essential oils, organic acids, prebiotics, phytochemicals, spices, and probiotics has been acknowledged and is currently suggested both for antibiotic replacement in farms and animal nourishment among smallholders (Ogbuewu et al., 2022).Such non-antibiotic feedstuff (herbs and additives) are being considered capable of bridging this

A Comparative Study of the Effect of Non-Antibiotic Feed Additives on Experimental Colonization of Salmonella Enterica Serovar Enteridis and Intestinal Pathomorphology in Broiler Chickens
gap, as some agronomists used to utilize these instead of antibiotics for poultry (Nava et al., 2005;Jong et al., 2020;Mustafa et al., 2021a;Abbas et al., 2022a;Mohamed et al., 2022).
Probiotics exhibit numerous significant modes of action, such as an antagonistic action against pathogenic microbes by altering gut pH; having an antimicrobial effect by the excretion of products inhibiting their expansion, such as bacteriocins, hydrogen peroxide, and organic acids; the intestinal production of short-chain fatty acids (SCFA); host immune system regulation; regularization of intestinal microbiota, along with diverse metabolic results (Vamanu et al., 2010;Ferreira et al., 2011).Favorable effects of probiotic addition may be seen in amplified abdominal enzyme creation, reduction of phenol and ammonia yields, and increased resistance against the pathogenic microbe's propagation in the abdomen by the mean of competitive exclusion (CE; Yusrizal & Chen, 2003).CE is a beneficial strategy consisting of adding specific probiotics (culture of non-pathogenic bacteria) to the gastrointestinal tract (GIT), which ultimately hinders the colonization of pathogenic bacteria, and produces subsequent competition for accessible nutrients and growth elements (Patterson & Burkholder, 2003;Konieczka et al., 2022).
Prebiotics are particular fermented components that allow specific variations in all the configurations and actions of the gastrointestinal microbiota and ultimately favor host health (FAO/ WHO, 2002).Prebiotics comprising galactose, glucose, mannose, fructose, and xylose has been largely studied and seem to be predominantly positive (Gibson & Roberfroid, 1995;Patterson & Burkholder, 2003).Most of them have been demonstrated to improve defense against Salmonella, since prebiotics physically imitate their binding sites and prevent their adhesion to intestinal epithelial cells (Ferreira et al., 2011).Essential oils, commonly known as ethereal or volatile oils, are aromatic greasy fluids obtained from plants.They have a favorable effect on the metabolism of lipids, anti-microbial and anti-oxidant characteristics, antiinflammatory properties, and work as digestive stimulants (Rota et al., 2004;Acamovic et al., 2005).Owing to their antimicrobial characteristics, essential oils are regarded as potential feed additives (Dibner & Buttin, 2002;Lee et al., 2004).Short-chain fatty acids (SCF) and medium-chain fatty acids (MCFA) in animal feed have been widely proclaimed as anti-microbial agents.Their anti-microbial characteristics are due to their ability to lower pH due to the dissociation of carboxyl groups, which can infiltrate bacterial cells and ultimately cause cell death.Due to their antimicrobial characteristics, organic acids are considered substitutes for synthetic growth promoters ( Cherrington et al., 1991;Dibner & Buttin, 2002).
Customer refusal of artificial food extracts and antimicrobial growth promoters (AGP) has increased due to the devastating stated effects of these harmful chemicals.Therefore, the present research focuses on the potential advantageous results of non-antibiotic feed additives for an improved understanding and awareness of the topic.

Source of birds
A total of 125 Hubbard classic A-grade (38.5 gm) day-old broiler chicks were procured commercially were raised in the experimental open-sided poultry house of the Pathology Department, University of Veterinary and Animal Sciences, Lahore.All bird handling procedures are in line with International Animal Care.

Housing Conditions
Washing of the shed and utensils was done with high-pressure water, followed by washing and scrubbing with surf and phenol, with subsequent sun drying.Later on, the shed was whitewashed with limestone, and formalin was sprayed on the walls and floor.Finally, fumigation was performed after putting in the utensils and litter.Chicks were housed in pens of identical size (1×2 m 2 ) in a deep litter system with rice husk.On the first day, the room temperature was set at 32 °C and lowered stepwise to 24°C for the rest of the experiment.Feed (pre-starter and starter) in the form of crumbs was purchased commercially and formulations were made according to NRC (1994) guidelines.
Birds were reared under the same management and environmental conditions.A LED light of 30 lx was used for 22 hours for the first 3 days, for 20 hours for the first week, and the duration and intensity were further reduced thereafter with the age of the birds.

Treatments
The probiotic used contained spores of Bacillus subtilis at cfu/g, and was mixed in the feed at the rate of 10 g /t.Prebiotic, a refined yeast-based mannan oligosaccharides preparation, was added at the rate of 400g/ton in the feed.Essential oils, a water-soluble concentrate containing essential oils of Eucalyptus, Menthol, and Saponins was added to drinking

A Comparative Study of the Effect of Non-Antibiotic Feed Additives on Experimental Colonization of Salmonella Enterica Serovar Enteridis and Intestinal Pathomorphology in Broiler Chickens
water at 0.25ml/liter.The organic acids used were a synergistic combination of formic, lactic, and propionic acid or their salts, and a surfactant.It was added to the drinking water at 0.5 ml/liter.The recommended vaccination schedule for broilers issued by the National Disease Control Committee of the Pakistan Poultry Association as of 24 th May 2016 was followed.Upon arrival, five chicks were randomly selected and checked for Salmonella (presence/absence) in the ceca-cecal tonsils and were found to be negative.Trade names of the products are not displayed due to the commercial impact of the results.

Bacterial Stain and culture conditions
The challenge organism used in all experiments was a poultry isolate of Salmonella enterica serovar Enteritidis (SE), tested against antiserum.SE was cultured in Tetrathionate broth.Post-incubation, bacterial cells were collected, reconstituted in saline, quantified by total viable count, and diluted to an approximate concentration of 4 × CFU per 0.25 milliliter (Prado-Rebolledo et al., 2017).Concentrations of the isolate were further verified by serial dilution and plating on Salmonella-Shigella agar for enumeration of the actual CFU used to challenge the chickens.

Experimental Design
The chicks were divided randomly into six groups (A, B, C, D, E, and F), with 20 chicks per group and two replicates in each group.The treatments were given to the respective groups after experimental infection upon arrival and were continued during the whole experiment.The treatments were as follows: Group A was given a basal diet (The negative control group).Group B Challenge + Basal diet (Positive control group).Group C: Probiotic + Challenge + Basal diet.Group D: Prebiotic + Challenge + Basal diet.Group E: Essential oils + Challenge + Basal diet.Group F: Organic acids + Challenge + Basal diet.Two separate experiments were carried out for Salmonella recovery by checking cecal tonsils and conducting an intestinal pathomorphic evaluation.Villus length, villus width, villus surface area, and crypt depth were measured by micrometry.

Experiment 1
This experiment involves evaluating Salmonella establishment and colonization in ceca-cecal tonsils.Groups B, C, D, E, and F were challenged with SE at 4 × CFU/0.25 mL per bird through oral gavage after arrival.Group A was given sterile normal saline as a vehicle through oral gavage.

Salmonella Recovery
Four birds were nominated indiscriminately from all the designed groups, seventy-two hours postchallenge, and were slaughtered humanely.Ceca-cecal tonsils were harvested aseptically after performing post-mortem.After harvesting the particular tissue, the ceca-cecal tonsils underwent homogenization and enrichment aseptically in tetrathionate broth.They were diluted with saline and serial dilutions postenrichment and were plated on Salmonella-Shigella agar to enumerate the cfu/g of ceca-cecal tonsils and to check for positive H 2 S, non-lactose fermenting, colorless clear transparent colonies with dark black mid-points to ascertain the presence or absence of Salmonella (Prado-Rebolledo et al., 2017).

Intestinal Morphology Evaluation
For the intestine morphometric analysis, at 72 hours, 7 days, and 14 days, two birds from all the groups were chosen randomly and slaughtered humanely.Two different intestinal sections of approximately 1cm, one from the lower ileum and the other from the center of the duodenum, were aseptically collected from each slaughtered bird, after carefully washing the tissue with normal saline for the removal of any intestinal content residues.Standards techniques routinely practiced in the UVAS pathology lab were used for the histopathological examinations of the collected tissue samples.The steps comprise the fixation of the tissue, dehydration of the sample, clearing, sectioning post embedding, and lastly the careful staining of the sectioned tissue (Athanassopoulou et al., 1999).

Intestinal Morphology Analysis
The stained slides were then examined under 4X magnification and pictures were taken and analyzed by PixelPro software by Labomed Inc. Villus length was measured from the top of the villus to the top of the lamina propria (Mustafa et al., 2021b).Crypt depth was measured from the base upward to the region of transition between the crypt and villus (Aptekmann et al., 2001).Villus width was measured at the widest area of each villus, while the villus : crypt ratio was determined as the ratio of villus height to crypt depth.Villus surface area was calculated using the formula (2π) (VW/2) (VL), where VW = villus width and VL = villus length (Sakamoto et al., 2000).

Statistical Analysis of the Data
The collected data from both experiments were statistically analyzed using a completely randomized design and means were compared by ANOVA (Analysis of variance) through SPSS 16.0.

RESULTS
The results of experiment 1 consist of the total number of birds found positive for Salmonella presence out of the total number of birds tested and the Log10 of Salmonella Enteritidis /g of cecacecal tonsils.72 hours post-challenge, four birds were selected randomly from each of the groups, humanely slaughtered, and checked for enumeration of Salmonella CFU/g.Results of this experiment are given as the total number of birds found positive for Salmonella presence out of the total number of birds tested, and the Log10 Salmonella Enteritidis/g of ceca-cecal tonsils (Table 1).The result indicates all birds were found positive in all treatments except the control group.Moreover, the lowest prevalence (Salmonella CFU/g ) was observed in groups C and F which were fed probiotic and organic acid, respectively.Experiment 2 consisted of an enteric morphometric analysis: two birds from each of the groups were humanely slaughtered on the designated evaluation day (3 rd day, 7 th day, and 14 th day).Segments of the midpoint of the duodenum and the distal end of the lower ileum from each bird were collected and processed for histopathological examination (Figures 1 & 2).For this experiment, results are given as the average value calculated from 5 sections of both the duodenum and ileum of 2 birds per group at the designated evaluation days.All values are expressed as Mean ± Standard deviation (Tables 2 & 3).On the 3rd day of the study, there was a significant difference in the mean of all the parameters of morphometric analysis of duodenal mucosa between all six groups.Group F, which was fed with organic acids, showed the highest villus height of 1060.4±87.76µm, crypt depth of 143.07±9.80µm, and villus surface area index of 477.72±64.94mm 2 in the duodenum.On the other hand, the result of iliac mucosa showed that the prebiotic group had improved villus height, crypt width, villus dept, and villus surface area index as compared to other groups.On the 7 th day, the result of duodenal mucosa indicated that group E had improved crypt width and villus surface area index.However, organic acids showed a pronounced effect on the iliac mucosa.

Effects of early feeding in combination with probiotics
There was an overall increase in all the parameters of intestinal morphometric analysis for all the treatment groups, except for the control negative group which showed the lowest values.The maximum villus height eRBCA-2022-1620 ac Values within columns with no common superscript differ significantly at p<0.05. of 1794.2±63.96µm in the duodenum was achieved by group E, which was fed essential oils, whereas the maximum villus surface area index of 1662.6±389.16mm 2 was recorded in group D, which was treated with prebiotics.The maximum villus height of 940.35±23.96µm and surface area index of 568.92±36.27mm 2 in the ileum mucosa were recorded in group D, treated with prebiotics.
Final results show that there is an overall increase in histological parameters of the mucosa of the duodenum and ileum in the groups fed non-antibiotic feed additives as compared with positive and negative controls.Prebiotics showed the maximum positive effects.Therefore, this study suggests that a combination of non-antibiotic feed additives will be beneficial for the intestinal health of broiler chickens, but there is a need for more research on the combinations of non-antibiotic feed addition.

DISCUSSION
Growth promoters are used in poultry feed to enhance the microflora of intestines and to develop the immune system to ultimately improve performance.However, antimicrobial agents as prophylactic means have been strongly questioned due to the advancement of antimicrobial resistance amidst pathogenic microorganisms.Consequently, the possibility of antibiotics not being used as progression growth drugs for poultry anymore and the apprehension about the secondary results of their consumption as therapeutic mediators has formed an atmosphere in which consumers and manufacturers are equally seeking alternatives.In the current study, treatment with probiotics improved the morphology of the intestine as described by a study performed by Biloni et al. (2013), in which a combination of Early Bird and FloraMax-B11 supplementation (an encouraging probiotic supplement) was used.Researchers deter-mined that a mix of Early Bird FloraMax enhanced gut morphology with the reduced recoverable amount of Salmonella, while also increasing poultry mass in comparison with controls separately by each product.Till today, a list of products comprising plant extracts, essential oils, organic acids, prebiotics, spices, and probiotics have been acknowledged and suggested equally for antibiotic substitutions in farms and smallholder animal nourishment.Such non-antibiotic feedstuff herbs and additives are being studied to bridge this gap, as some agronomists use to utilize these instead of antibiotics for poultry (Griggs & Jacob, 2005;Nava et al., 2005).The final results of this study show an increase in intestinal health of broiler chicks that were treated with non-antibiotic feed additives in the presence of the challenge strain Salmonella enterica serovar Enteritidis.There is an overall improvement in intestinal health as measured through the micrometry technique.Villus height, villus width, crypt depth, and villus surface area index increased in groups given non-antibiotic feed additives treatments such as probiotics, prebiotics, essential oils, and organic acids (Abbas et al., 2022b).The results of prebiotics on the ileum mucosa were the highest, and essential oils and prebiotics both contributed to the highest results for the improvement in duodenal morphology.When compared with a negative control group and a positive control group, all the non-antibiotics showed gut morphology improvement.In this trial, the increase in gut morphology by treating with prebiotics can be supported by a study conducted by Sultan et al. (2015) who analyzed the effects of a particular strain of yeast-derived-carbohydrate-fractions (Actigen) at various stages on the performance of the broiler and gut histo-morphology.
The overall improvement in gut morphology caused by non-antibiotic feed additives shows the need for these products to be used in combination rather than alone.Such combinations may help farmers to overcome the issue of a ban on antibiotics and antibiotic resistance.

CONCLUSION
This experiment aimed to evaluate the antimicrobial and growth promoter efficiency of probiotics, prebiotics, synbiotics, essential oils, and organic acids.These non-antibiotic antimicrobial agents have gained popularity due to their positive effects on growth, gut health, metabolism, and immunity.Dietary use of prebiotics and probiotics is a pre-requisite for regulating the micro-flora to promote better health and prevent diseases.Based upon the results of the present research, it can be concluded that the dietary addition of nonantibiotic antimicrobial chemicals such as probiotics, prebiotics, organic acids, and short-chain fatty acids in poultry feed may have good and safe antibiotic effects without having any residual or side effects on body organs, especially gut histomorphology.However, further research is required to clearly understand the mechanism of action of these chemicals at the cellular level.

STATEMENT OF ANIMAL RIGHTS
All animals were handled according to international, national, and institutional guidelines for the care and use of animals.

Table 1 -
Results of this experiment are given as the total number of birds found positive for Salmonella presence out of the total number of birds tested, and the Log10 Salmonella Enteritidis /g of ceca-cecal tonsils at day 3.