An updated review on probiotics as an alternative of antibiotics in poultry — A review

Antibiotics used to be supplemented to animal feeds as growth promoter and as an effective strategy to reduce the burden of pathogenic bacteria present in the gastro-intestinal tract. However, in-feed antibiotics also kill bacteria that may be beneficial to the animal. Secondly, unrestricted use of antibiotics enhanced the antibiotic resistance in pathogenic bacteria. To overcome above problems, scientists are taking a great deal of measures to develop alternatives of antibiotics. There is convincing evidence that probiotics could replace in-feed antibiotics in poultry production. Because they have beneficial effects on growth performance, meat quality, bone health and eggshell quality in poultry. Better immune responses, healthier intestinal microflora and morphology which help the birds to resist against disease attack were also identified with the supplementation of probiotics. Probiotics establish cross-feeding between different bacterial strains of gut ecosystem and reduce the blood cholesterol level via bile salt hydrolase activity. The action mode of probiotics was also updated according to recently published literatures, i.e antimicrobial substances generation or toxin reduction. This comprehensive review of probiotics is aimed to highlight the beneficial effects of probiotics as a potential alternative strategy to replace the antibiotics in poultry.


INTRODUCTION
Since they were discovered antibiotics (ABs) have been used for prevention and treatment of infectious diseases and when added at sub-therapeutic level in diet as a growth promoter in many animal species. Antibiotics also performed significant role in the improvement and success of animal husbandry. Chicken is the only species with highest population worldwide reared as food animal and highest concentration with different strains, ABs are used in poultry farming [1]. When ABs are used in any system, they kill the susceptible bacterial leaving behind some resistant strains, which increase in number with the passage of time and might become the cause of resistant genes transfer to other bacteria [2]. These resistant bacteria may transfer from one host to other, directly, or indirectly and lower the effectiveness of drugs and become the cause of antibiotics resistance (ABR), [3]. In this way ABs exhibit negative effects on animals and human when used continuously at subtherapeutic for long time or used in excess [4]. ABR in bacteria could ether be inherent directly or acquired from other species of bacteria in the environment [5] and develops by different ways such as lowering the permeability of bacterial cell membrane, developing changes in binding sites where the ABs attach in bacteria or/and alteration in enzyme production [6]. There are several bacteria which have developed ABR due to improper use of ABs. Staphylococcus species which commonly affect the chicken and cause pododer-

CONCEPT OF PROBIOTICS, COMMON VARIETIES AND SELECTION
"Probiotics could be mono or mixed cultures of the live microorganisms which have some beneficial effects on the host when fed to them in adequate amount" [16]. Angelakis, [19] stated some characteristics of a good probiotic i.e., it should not be pathogenic or toxic in nature, should have positive impact on host animal, should survive in gut environment, should be viable and remain viable under stored condition. Bifidobacterium, Bacillus, Streptococcus, Lactobacillusa and Lactococcus are the genera commonly used as probiotics in poultry [20]. Other commonly used probiotics are L. casei, B. subtilis, L. acidophilus, L. bulgaricus, L. plantarum, L. lactis, Enterococcus faecalis, E. faecium, Bifidobacterium spp., Saccharomyces cerevisiae (S. cerevisiae), and Aspergillus oryzae. Much work has been done on Bifidobacterium spp. in humans as well. Enterococcus spp., Bacillus spp., and Saccharomyces spp. are generally used probiotics in livestock [21]. Probiotics could be a single or multi strains and may be given in combination with other feed additives through feed or water. It is generally accepted that multiple strains probiotics are more beneficial than single stain because multiple strains have synergistic effects [22]. Probiotics exist in different forms such as granules, power, liquid, paste and gel etc. Dry form is better for storage and gastric environment [23]. Selection criteria for probiotics includes following characteristics: survival under gastrointestinal conditions, competitive exclusion of pathogenic bacteria, ability to stay/attach to mucosa of gastrointestinal tract [20] and ability to survive under different feed processing conditions [24].

PROBIOTICS ON GROWTH PERFORMANCE AND MEAT QUALITY
Though the mechanism of probiotics to improve the growth performance is not fully understood, studies suggested that they are growth enhancers (Table 1) and ameliorate the effects of disease attack and stress. Growth performance of broilers is improved by using single and multiple strains of Lactobacillus spp. [25][26][27]. Pediococcus acidilactici (P. acidilactici) significantly improved the feed intake, weight gain (WG), and feed conversion ratio (FCR) in broilers [28]. Significant increase in WG was detected using different types of probiotics [29,30]. L. fermentum was reported to have various results in combination with different probiotics, no significant effect was observed with S. cerevisiae [31] and slight increase in body weight of broilers with E. faecium [32]. B. subtilis and E. faecium significantly improved final body WG and FCR [33,34]. Different strains of B. subtilis alone or in combination of E. faecium significantly improved the growth performance of broilers and layer [33,35,36]. However, contradictory results were found by using Lactobacillus spp. (L. crispatus, L. ohnsonii, L. salivarius, and some unidentified Lactobacillus spp.), [37]. Feeding multiple strains of probiotics (E. faecium, Bifidobacterium animals, P. acidilactici, L. reuteri, and L. salivarius) significantly improved WG, FCR, and production efficiency factor [38]. Many meta-analyses have also been done to evaluate the effect of probiotics on broilers and other animals. A meta-analysis was conducted by Faria-Filho et al [39] to observe the response of broilers by feeding 12 different probiotics. In this analysis, data was collected form 27 studies involving 30,146 broilers. They concluded that WG and FCR was improved by probiotics feeding. A more comprehensive meta-analysis comprising studies of 32 years were performed by Blajman et al [40]. They stated that probiotics feeding had positive effect on WG and FCR. They also suggested no difference in using single or multi-strain probiotics, but probiotics provided in water were more effective than via feed. Depending upon the health status, boilers respond differently to different probiotics. Immune modulation and balancing of gut microbiota are two possible reasons for improving growth performance of broilers by probiotics feeding. Probiotics prevent the proliferation of pathogenic bacteria by decreasing the intestinal pH through production of short chain fatty acids and maintain the equilibrium between beneficial and pathogenic bacteria which is essentially required of gut health and proper growth performance of broilers.
Variable results are found in literature about the effect of probiotics on meat quality in broilers. Improvement in meat flavor was observed by feeding broilers on B. subtilis and B. licheniformis [41] however, no significant effect of probiotics was found on sensory attributes of meat in another study [42]. Mahajan et al [43] stated that meat sensory characteristics (overall acceptability, texture, appearance, and juiciness) were positively affected by probiotics feeding. In addition, Pelicano et al [44] stated that probiotics feeding improved the meat flavor. Previous results were also supported by Ceslovas et al [45], they found improved meat quality and tenderness by probiotics. Contradictions in the results might be due to use of different type or dose of probiotics, duration of experiment or age of birds at which the probiotics were fed to the birds.

PROBIOTICS ON BONE HEALTH AND EGGSHELL QUALITY
Previous studies confirmed that probiotics supplementation also has positive effects on bone health in poultry. Probiotics improved the gut health, enhanced the absorption capacity of intestine and bioavailability of minerals by different ways.
Most of the studies presented the effect of various probiotics on tibia bone, might be since tibia is more prone to skeletal problems. Bacillus subtilis improved the tibia ash and ash Ca percentage [46]; Enterococcus faecium and lactic acid producing bacteria improved different tibia indexes including tibia Ca and Ca percentage [47]; B. subtilis and B. licheniformis improved tibia thickness, ash and P percentage [48]; Lactobacillus sporogenes enhanced tibia breaking strength and ash percentage [49] whereas Clostridium butyricum and B. subtilis also enhanced tibia breaking strength, ash contents and tibia weight/length index [50] in broilers. Additionally, probiotics also have the ability to protect the bone under different pathological conditions as described by Sadeghi [46], that supplementation of B. subtilis in broilers challenged with Salmonella enteritidis (S. enteritidis) improved the tibia bone minerals contents including Ca at 21 days of age. Probiotics also reduce the negative effects of low Ca diet in broilers [50].
Another study indicated that supplementation of B. subtilis in diet of laying hens (at 0.5 or 1.0 g/kg of feed) boosted the tibia indexes such as length, weight, density, and ash contents [51]. Similarly, B. subtilis feeding in laying hens also decreased the number of unmarketable eggs [52]. Furthermore, different probiotics also improved the eggshell quality in laying hens [53]. Basic reason underlying improved bone health by probi-otics feeding is enhanced Ca absorption by different ways (Figure 1). An in vitro study reported that L. salivarius enhanced trans-epithelial Ca transport in human cells, while in vivo study supported this result as Ca and P concentration was higher in probiotics supplemented rats than control group [54]. Probiotics increase Ca bioavailability through different ways such as they produce phytase having ability to degrade phytate and release bounded minerals like Ca and P [55]. As mentioned in another section that probiotics positively affect the gut morphology and increase the absorption area of intestine by increasing villus length and villus length to crypt depth ratio. So, higher absorption area increases the rate of minerals absorption. Another possible way of increasing mineral absorption is reduced intestinal pH due to short chain fatty acids produced by probiotics. Acidic environments favor the absorption of Ca [56].

PROBIOTICS ON IMMUNE RESPONSE
Probiotics as an alternative to ABs are considered to improve the health status and immunity in poultry birds (Table 2). Bai et al [31] demonstrated that T cell immune system was improved by probiotics (combined: L. fermentum and S. cerevisiae) without compromising the growth performance of broilers. It is also proved that Lactobacillus (LAB) influenced the chemokine gene expression and cytokines production in chicken [57]. LAB improved the production of anti-and pro-inflammatory cytokines in the intestinal epithelium of broiler chicken. The cytokines production influences the overall immune system. Increase in antibody production by β-lymphocytes is another potential mechanism of LAB in improving the immunity in broiler. L. fermentum improved the blood CD4 + lymphocyte value as well as interferon-γ and tumor necrosis factor-α expression in the ileum. Waititu et al [58] fed a mixture of three Bacillus subtilis strains (BSS) and Propionibacterium acidipropionici (PA) to broilers and found downregulation of ileal expression of different cytokines by PA and interleukin-10 (IL-10) in the spleen, IL-2,  IL-4, IL-6, and toll like receptor-2b in all examined tissues by BSS, whereas IL-13 was upregulated in spleen by BSS. Kabir et al [30] found significant increase in the antibodies concentration and weight of immune organs (bursa and spleen) of broilers by using Protexin Boost (a mixture of different probiotics) at the rate of 0.2 g/liters of drinking water.
E. faecium improved immunoglobulin A production in layers [59]. Similarly, findings of Haghighi et al [60] also suggested the enhanced systemic immunity and local antibodies production in broilers at 14 days post hatching by feeding LAB in combination with B. bifidum and S. faecalis. LAB also increase intestinal intraepithelial lymphocyte expression of the surface markers CD3 + , CD4 + , and CD8 + in broilers [61]. L. fermentum (10 7 cfu/g) with S. cerevisiae (2× 10 6 cfu/g) significantly increased CD8 + , CD3 + , and CD4 + levels in broilers [31]. Hatab et al [33] found improved antibody production against Newcastle disease virus in layers birds by B. subtilis and E. faecium. Similarly, B. subtilis feed-ing increased the antibody titers levels in broilers against Newcastle disease, Infectious bronchitis, and Infectious bursal disease virus [35].

PROBIOTICS ON INTESTINAL MICROBIOFLORA AND MORPHOLOGY
Animal health status and growth performance are directly linked with gut health and its microflora. A healthy gut is more defensive against pathogenic microorganisms and works more efficiently for nutrients absorption. A stable microflora is also required to avoid infections in the gut which helps the animals in different ways, by avoiding the colonization of pathogens by bacterial antagonism or occupying the attachment site in gut and interfering with bacterial activities. The intestinal microflora is quite stable but influenced by different environmental factors and health status of animals. Environmental factors include diet, hygiene conditions and stress. The most important factor which affects the gut microflora is diet. Probiotics are generally applied to regulate the gut microbiota [62] (Figure 2). Similarly, gut morphological parameters also have significant importance towards health and performance of poultry birds and animals as higher villus length to crypt depth ratio with higher villus height is directly linked to increased absorption area and nutrient absorption.
In addition, goblet cell concentration in intestinal villi is another gut health parameter because these cells reduce the chances of attachment of pathogenic bacteria to intestinal epithelium and enhance mucin production [63]. Probiotics have positive effects on gut histomorphology, but the degree of effectiveness may vary from strain to strain. As increase in villus height with decrease in crypt depth was observed in broilers fed probiotic containing L. acidophilus, L. casei, Enterococcus faecium, and Bifidobacterium thermophilum [17]. Similarly, supplementation of B. coagulans [64]; combination of L. acidophilus, L. casei, Enterococcus faecium and Bifidobacterium thermophilum, [65]; L. reuteri and L. salivarius [66]; Propionibacterium acidipropionici [67]; mixture of B. licheniformis, B. subtilis and S. cerevisiae [68] and P. acidilactici [69] in broilers exhibited positive effect on gut histomorphology with increased villus length and villus length to crypt depth ration which suggested that probiotics enhanced nutrient absorption. Hayashi et al [70] fed B. subtilis to broilers and found improved histologic modification associated with stimulation of the defense reaction in the ileum. Most of the LAB species affect the gut health positively. Chen et al [71] found significant reduction in Salmonella counts recovered from the cecal tonsils, spleens, and livers and Gao et al [25] observed accelerated maturation of intestinal microbiota by LAB feeding in broilers. LAB may be efficacious for reduction of S. enteritidis in young chicken [72]. Increased anaerobic bacteria in the ileum and caeca, and lactic acid bacteria and lactobacilli in the caeca with significant increase in weight of small intestinal (jejunum and ileum) was observed with LAB. Probiotics also reduced the count of pathogenic bacterial [37]. Palamidi et al [38] suggested that LAB increased the digestibility of dry matter and fat in diet. LAB significantly increased the number of beneficial bacteria one-week post feeding and reduced the count of pathogenic bacterial by 40 days post  feeding in broilers [27]. It is suggested by researches that probiotic feeding increase the total count of beneficial bacteria in intestine and reduce the number of disease-causing bacteria [73,74]. Supplementation of B. subtilis alone or in combination with S. boulardii have a significantly positive effect on intestinal histopathology and microflora in broilers [34,35] (Table 3).

UPDATED ACTION MODE OF PROBIOTICS
There are different mechanisms through which probiotics work to facilitate the host animals, which may include occupation of epithelial cells to avoid the colonization of pathogenic bacteria, stimulation of enzyme synthesis, production of some antimicrobial substances and reduction of toxin production, boost up the immunity and stress reduction [75] ( Figure 3). Occupation of the site of attachment in GIT by probiotics to avoid pathogenic bacteria could result in physical blocking of pathogen colonization. This physical blocking alters the environment of GIT and is beneficial for host animals to improve their immune system. This mechanism was found by Nurmi and Rantala [76], suggesting that Salmonella colonization in the GIT of newly hatched chicken could be reduced by feeding a suspension of gut contents of healthy adult chicken. In this way pathogenic bacteria are excluded from the site of replication. Probiotics also produce some antimicrobial sub- stances like bacteriocins, hydrogen peroxide and organic acids [77]. Organic acids decrease the pH of intestine which helps absorption of minerals (calcium, iron, copper, magnesium, and manganese) and protein. Probiotics help to regenerate intestinal mucosa, upregulate the mucous production and intestinal motility, modulate host immune system by stimulation of antibody production and natural killer cells [77], and improve the digestion by increasing the secretions of digestive enzymes. Probiotics can also reduce the blood cholesterol level by virtue of their bile salt hydrolase activity which lower the chances of cardiovascular disease [78]. There is another mode of action in probiotics called cross feeding, which means feeding of one bacteria is beneficial for another bacteria production. For example, lactic acid produced by lactic acid producing bacteria can be used by butyric acid producing bacteria and produce a large amount of butyric acid [79]. The cross-feeding mechanism of butyric acid production is beneficial to enhance the growth performance.

CONCLUSION
Keeping in view the past studies, it could be concluded that probiotics could be serve as growth promoters because they improve the gut health which ultimately improves the nutri-ent digestibility by enhancing the digestive enzyme activities and improves the growth performance and meat quality. Probiotics also modulate the immune response in such a way to protect host animals from pathogenic disease, in addition through competitive exclusion mechanism they protect the host animals from colonization by pathogenic bacteria. Inconsistency in results could be due to numerous biotic and abiotic factors which could be resolved by further studies concerning detail of mode of action, mode of delivery and improving probiotic in vivo efficacy.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.  Showing mode of action of probiotics that results improved production performance in monogastric animals by producing many gut microbiota modulating factors (like bacteriocins, organic acids etc) that improve immunity, reduce stress, improve nutrient production and utilization and modulate competitive adhesion to gut.

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526 Figure 2. Showing mode of action of probiotics that results improved production performance in 527 monogastric animals by producing many gut microbiota modulating factors (like bacteriocins, 528 organic acids etc) that improve immunity, reduce stress, improve nutrient production and 529 utilization and modulate competitive adhesion to gut. 530 531 532 533