Potential Substitutes of Antibiotics for Swine and Poultry Production

Abstract

Early of the last century, it was detected that antibiotics added to the animal feeds at low doses and for a long time can improve technical performances such as average daily gain and gain-to-feed ratio. Since then, the antibiotics have been used worldwide as feed additives for many decades. At the end of the twentieth century, the consequences of the uses of antibiotics in animal feeds as growth promoters were informed. Since then, many research studies have been done to find other solutions to replace partly or fully to antibiotic as growth promoters (AGPs). Many achievements in finding alternatives to AGPs in which probiotics and direct-fed microorganism, prebiotics, organic acids and their salts, feed enzymes, bacteriophages, herbs, spices, and other plant extractives (phytogenics), mineral and essential oils are included.

1. Introduction

The antimicrobial growth promoters (AGPs) or antibiotics have been used in animals at treatment/sub-therapeutic concentrations for enhancing the productivity and preventing diseases. The beneficial effects of using antibiotics were first advocated by Moore et al. when they found that chicken fed streptomycin with adequate amounts of folic acid exhibited increased growth responses [1]. Later, in 1951, the US Food and Drug Administration (FDA) approved the use of AGPs in animal feed without veterinary prescription [2]. Subsequently, the use of AGPs has become globally practiced, significant rising by 10–20-fold since 1950s.

Despite the positive effects of AGPs being well documented, their use was also controversially argued for a long time due to the risks of antimicrobial resistance, posing a potential threat to human health [3]. For instances, the resistance to antibiotics has been increasingly observed since the first cases of streptomycin resistance in food animals were recognized in 1951. In fact, there is irrefutable evidence that foods from many animal sources and all food processing stages contain a large number of resistant bacteria [4]. Consequently, there are approximately 23,000 and 25,000 deaths annually occurred due to the antibiotics resistance in the USA and Europe, respectively [5, 6]. Antimicrobial resistance had led to the failure of treatment in 195,763 cases of pneumococcal disease, which contributed to 2925 child deaths annually in Ethiopia [7].

Since the big concern of antimicrobial resistance to global public health, the European Union issued a ban all AGPs on precautionary grounds in 2006 [8, 9]. In the World Health Day 2011, the subject “combating drug resistance: no action today, no cure tomorrow” was discussed to reinforce all countries in the whole world to take proactive actions against antimicrobial resistance. Furthermore, The World Health Assembly 2015 approved global action plan to tackle the issue of bacterial resistance. In addition, the 2016 United Nations High-Level Meeting on antimicrobial resistance and the G20 Summit in Hangzhou, China, launched strong commitments to control the crisis of antimicrobial resistance [10].

Both political and consumer pressures are prompting a reduction in the use of AGPs in animal production; therefore, the identification of alternatives might be reasonable approach that may help reduce the risks and prevent the spread of drug-resistant bacteria and may promote the animal breeding industry. A great deal of studies have focused on the development of alternatives to AGPs including probiotics, prebiotics, synbiotic, enzymes, phytogenics, antimicrobial peptides, bacteriophage, and antibody therapy [3, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20]. In addition, in recent years, the CRISRP-Cas9 gene editing tool showed effective impacts on preventing infectious diseases that is a promising approach to alternative to AGPs in the future [21]. This review, therefore, focuses on such alternatives along with a description of their efficacy in swine and poultry production.

2. Modes of action of AGPs

It is believed that the successful development of antibiotic alternatives relies on understanding the mechanism of action of AGPs. Current evidence shows that at least two major modes have been proposed to explain the function of AGPs: (i) bacteria-centric and (ii) host-centric. Further, other environmental factors including stress, diet, and nutrition will influence both the host and the microbiota, and interactions among these factors with AGPs are also important to their function [22].

2.1 Bacteria-centric mode of action

The intestinal mucosa is central both to nutrient absorption and to maintaining the immunological homeostasis, and the complex microbial communities that harbors gastrointestinal tract (GIT) are involved in the host immunological and metabolic processes [23]. AGPs are utilized as feed additives, and thus, the GIT is considered as a primary site of AGPs action. The initial study on germ-free mice with AGP administration did not exhibit the increased growth suggested that modulation of the intestinal microbiota is central to the action mechanism of AGPs [24]. The growth-promotion phenotype was shown to be transferrable to germ-free hosts by low-dose antibiotic-selected microbiota, indicating that the altered microbiota and not the antibiotics played a causal role [25]. It was also shown from the studies in mice that exposure to low-dose antibiotics early in life induces long-term host metabolic effects by accelerating normal age-related microbiota development and altering ideal expression of the genes involved in immunity [26]. Bacteria-centric hypotheses propose that AGP-induced changes to bacterial communities lead to enhanced growth by modulating the microbiota to create a more efficient system. This may include altering competition for nutrients, preventing pathogen colonization, and/or selecting for bacteria that are able to extract more energy from the diet [22].

2.2 Host-centric modes of action

The intestine not only plays vital roles in nutrient absorption but is also a major immunologically responsive organ. The “host-centric hypothesis” is supported by evidence that several antimicrobial agents have immune-modulating properties at therapeutic concentrations, which include downregulation of prolonged inflammation, increased mucous clearance, and modified phagocyte activity [22]. The direct effects of AGPs on the host physiology were indicated by Brown et al. [27]. They found that Altered Schaedler Flora (ASF) mice treated with chlortetracycline or tyrosine phosphate had lower expression of βd1 and Il17a in the intestine and had a strong induction of Il17a and Il10. In addition, by treating with AGPs, mice exhibited a lower hepatic expression of acute-phase proteins (Saa1, Hp, and Cp) in the liver tissue and Citrobacter rodentium-induced reductions in the expression of genes involved in lipogenesis (Hmgcl and Fabp1) [27]. Although the effects observed in mice cannot be directly extrapolated to farm animals, they might provide an insight into a possible mechanism of action and highlight important considerations in the development of alternatives to AGPs.

3. Alternative approaches to AGPs

3.1 Using available alternative compounds in the absence of AGPs

An ideal alternative approach should have the same beneficial effects of AGPs, enhancing the growth and preventing the diseases. Considering the proposal AGP mechanisms of action, the practical alternative compounds should possess the properties of modulating the gut microbiome and immune responses. Over the past decades, scientists have investigated and evaluated a variety of alternatives for antibiotics to control health issues related to animal production including probiotics, prebiotics, synbiotics, organic acids, enzymes, phytogenics, and trace minerals. The recently discovered novel alternatives such as hyperimmune egg yolk IgY, antimicrobial peptides (AMPs), and bacteriophages therapy have been developed.

3.1.1 Probiotics

FAO and WHO [28] defined probiotics as live microorganisms (yeast, fungi, and bacteria), which, when supplemented in adequate amounts, affect the host intestinal microbial ecosystem and may help prevent the growth of pathogens resulting in improved health and prolonged life. A multitude of studies on useful microorganisms for probiotics were performed and led to the successful development of commercial probiotic products as food supplements for humans or feed additives for farm animals [29]. Commercial strains of probiotics are commonly isolated from the intestinal microflora and selected based on the criteria including resistance to stomach acids and bile salts, ability to colonize the intestine or antagonize potentially pathogenic microorganisms [30]. The probiotics genera are mainly Bacillus, Lactobacillus, Bifidobacterium, Enterococcus, Pediococcus, and Streptococcus [31]. Further, other microbes such as Saccharomyces, Aspergillus oryzae, and many more along with their products are also classified as probiotics.

The use of probiotics in poultry production had considerably positive effects on productivity and health. A numerous of scientific studies have quantified the efficacy of probiotics for growth promotion in both broilers and layers [32, 33, 34, 35, 36, 37]. For examples, Huang et al. reported that Lactobacillus strains, Lactobacillus casei (low dose) and Lactobacillus acidophilus (high dose), and S. acidophilum (high dose), a strain of fungus, were able to promote the growth of broiler chickens [35]. Fesseha et al. recently revealed that Lactobacillus paracaseis sparacasei and Lactobacillus rhamnosus were beneficial for the growth performance by improving body weight gain (BWG), feed conversion ratio (FCR), feed intake (FI), and positively affects the growth of the chicken [36]. In addition to the improved growth performance, the use of probiotics was also shown to enhance the general immune function of broilers, modulate the intestinal microbiota, and increase the number of beneficial bacteria [38, 39, 40, 41, 42]. Park et al. indicated that the dietary B. subtitis supplementation enhanced growth, intestinal immunity, and epithelial barrier integrity when chicken were infected with E. maxima [42].

The use of probiotics for health and swine production has been widely reported in the literature. As early as in 1970s, some studies showed that the Lactobacillus supplements improved average daily gain (ADG) and FCR in starter pigs [43, 44]. Huang et al. demonstrated that dietary Lactobacilli supplementation improved ADG and average daily feed intake (ADFI) of the weaning pigs [45]. Le Bon et al. recently reported that using probiotics had positive effects on FCR of weaned pigs, the E. coli counts in the gut were reduced dramatically when compared with the non-treated pigs [46]. More studies of probiotic effects on the growth performance of pigs, including suckling, weanling, growing, and finishing pigs, have been reviewed in detail [47]. In addition to the growth performance, studies on the effects of probiotics on the reproductive performance of swine are also reported. Alexopoulos et al. reported that the sows fed BioPlus 2B (containing Bacillus licheniformis and B. subtilis) at a dose of 400 g/ton during the interval from 2 weeks prior to the expected farrowing date up to the weaning day improved the subsequent fertility, reduced piglet diarrhea, reduced pre-weaning mortality, and increased piglet body weight at weaning [48]. Ahasan et al. summarized the results of previous studies and showed that some probiotic species including Bacillus and Streptococcus improved the litter size and vitality, colostrum quality, milk quality, and quantity [29]. Moreover, the supplementation of probiotics was also shown to enhance the immune responses in swine. By using in vitro model for studying the interaction between microorganisms and the host, Liu et al. found that the L. acidophilus or L. rhamnosus GG treatment of the cells did not reduce the replication of porcine rotavirus, but the L. rhamnosus GG alone treatment post-rotavirus infection reduced the mucin secretion response induced by the virus. The L. acidophilus treatment prior to the virus infection increased the interleukin 6 (IL-6) response to the infection, whereas the L. rhamnosus GG treatment post-rotavirus infection downregulated the IL-6 response [49]. This beneficial effect in turn can lead pigs with better capacity of nutrient digestion and absorption, and better nutrient utilization and production performance [31]. Various studies demonstrated that supplement of probiotics such as Lactobacillus fermentum; Lactobacillus reuteri, and L. plantarum complex can improve the digestibility of dry matter, organic matter, energy, crude protein, crude fiber, and phosphorus compared with those with non-probiotic-treated pigs [50, 51, 52].

Generally, the sub-therapeutic use of antibiotics to improve growth and efficiency of farm animal production has been restricted or banned in more than 30 countries, but the application of AGPs in feed to prevent diseases and improve production performance of pigs as well as poultry is still a common practice in other parts of the world. Thus, the replacement of AGP with probiotics, to address the issue of antibiotic resistance, is very critical for public health and the global poultry and swine production.

3.1.2 Prebiotics and synbiotics

Among feed additives that have been studied as alternatives to AGPs, prebiotics have been exploited and applied broadly into swine and broiler diets in the recent decades. Gibson and Roberfroid defined prebiotics as “non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon, and thus attempt to improve host health” [53]. The dominant prebiotics are mannan oligosaccharides (MOS), fructo-oligosaccharides (FOS), raffinose, resistant starch, and resistant dextrins.

In broiler production, Rehman et al. reported that the dietary supplementation of MOS significantly improved BWG and FCR in both starter and finisher phases, and antibody titer for infectious bursal disease was improved by the interaction effect between probiotics and prebiotics, when compared with the control group [33]. Similarly, the effect of MOS from one commercial product on growth rate, gut health, and control pathogen colonization of broilers under Clostridium perfringens (C. perfringens) challenge was indicated. The results showed that FCR and BWG in broiler group treated with MOS were significantly better than the control group, and MOS level of 0.05% was enough to achieve a response competitive with that of the antibiotic. Other studies also indicated that supplementation of MOS from 0.04 to 0.08% could alter cecal microbial community composition by increasing the genus Bacteroides and decreasing the counts of coliforms and C. perfringens [13, 54, 55]. In addition to its effects on cecal microbiota, MOS also improved microbial community in other sections of the intestine, including the jejunum, the ileum, the jejunal mucosa, and the ileal mucosa [56, 57]. Similar to MOS, FOS, which is derived from plants, has also been reported to enhance performance and modulate the gut microbiota in broiler chickens [58, 59, 60].

In the swine production, dietary supplement of prebiotics such as MOS (0.04–0.08%) increased the growth performance of sows and piglets and modulated the composition of the swine gut microbiome [17, 61]. Similarity, Zivkovic et al. indicated that introduction of prebiotics (MOS and FOS) in the diets had positive effects on performances of sows and suckling piglets such as greater FI in lactating sows by 13.75%, more born piglets by 14.7% and heavier by 3.6% at birth, greater body weight of litter by 3.1% at weaning, better FI of pre-starter by 6.7% per litter during lactation [62].

There are also abundant studies indicating the beneficial effects of combination of probiotics and prebiotics termed as synbiotics. The use of synbiotics was based on the concept that a mixture of probiotics and prebiotics affects the host by improving the survival and implantation of probiotic organisms and by selectively promoting the growth or metabolism of beneficial bacteria in the intestinal tract [63]. Supplementation of diets with a synbiotic product was shown to significantly improve body weight, average daily gain, feed efficiency, and carcass yield percentage compared with controls or probiotic-fed broilers [64, 65, 66, 67]. There is a great potential for synbiotics to be used as antibiotic alternatives for improving performance and reducing pathogenic load in the intestines of poultry. However, when combined utilization of various probiotics and prebiotics to be used as synbiotics, it is critical to carefully consider and research trials should be conducted to investigate their synergistic effect compared with the use of either product alone.

3.1.3 Organic acids

Dietary supplement of organic acids has been considered as potential alternatives to AGPs, owing to their antibacterial nature [3]. For examples, Dibner and Buttin showed that organic acids can stimulate pancreatic juice secretion and increase villi height [68]. Fascina et al. reported organic acids enhanced immune responses and intestinal quality of broilers [69]. Ao et al. claimed that organic acids can reduce gut pH, thereby suppressing pathogenic bacteria growth, which in turn enhances gut health and nutrient intake [70]. Therefore, organic acids have been widely used and shown to have significant benefits in swine and poultry production over the years.

Most of organic acids used as feed additives can be described as volatile short-chain fatty acids (e.g., fumaric, acetic, propionic, lactic, butyric acids), medium-chain fatty acids (MCFA), and long-chain fatty acids (LCFA) [71]. Adil et al. indicated that birds fed diets supplemented with different organic acids including butyric, fumaric, and lactic acid showed significantly higher BWG and FCR [72]. Maximum improvement was achieved in the group fed 3% fumaric acid in the diet. Addition of organic acids in broiler diets also increased the villus height in all the segments of small intestines [72]. Ma et al. recently reported the effect of administered levels of organic acids on intestinal health, enzyme activity, and antioxidative characteristics in broilers. The results showed an increased concentration of IgA, D-lactate, and IL-10 in the serum of broilers diets with 6000 mg/kg mixture of organic acids. Dietary of 3000 mg/kg mixed organic acid decreased the pH value of duodenum and enhanced the amylase activity of the pancreas, the tight junction protein (mainly Claudin-1, Claudin-2, and ZO-1) in the duodenum of broilers. Also, the modulated structure microbiota and the reduced abundance of E. coli were observed in birds fed with both high and low level of organic acids [73].

In swine, use of organic acids has demonstrated efficacy as AGPs and has positive impacts on disease prevention [74, 75]. For examples, Upadhaya et al. reported that the supplementation of organic acids mixture (10% malic, 13% citric, and 17% fumaric acids) to the diet of growing and finishing pigs improved the growth, digestibility of dry matter, N, and energy, decreased E. coli counts, and increased Lactobacillus counts [76, 77]. Likewise, Ahmed et al. indicated that weaned piglets fed the diet with 0.4% organic acid mixture (4.1% propionic, 9.5% phosphoric, 10.2% lactic, and 17.2% formic acids) and diet with 0.5% pure citric acid increased Bacilli and Lactobacilli counts and reduced E. coli and Samonella counts [78]. Li et al. also reported that weaned pig fed diet with 0.2% organic acid mixture (butyrate, MCFA, phenolics) and diet with 0.3% short-chain fatty acid plus MCFA improved the gut health and showed a similar growth-promoting effect as antibiotics [79]. Hong et al. also reported that dietary with a blend of MCFA (caprylic and caproic acids) improved the performance and nutrient digestibility in weaned pigs [80].

3.1.4 Exogenous enzymes

The use of in-feed enzymes for replacement of AGPs in swine and poultry production has been proposed for the past decades. The potentials of using enzymes are to improve the digestive and absorptive function of the gut, which can allow the host to absorb nutrients to a greater extent [81, 82]. Further, the use of exogenous enzymes may improve the integrity of intestinal mucin, increase gastric residency of feed, and reduce inflammatory responses and other beneficial effects on immune function and resilience [83]. Generally, the enzyme systems available for animal feed are derived from microbes (fungi and bacteria) through the fermentation or genetic engineering. The main classes of enzymes include phytase, carbohydrase (xylanase, cellulase, α-galactosidase, β-mannanase, α-amylase, and pectinase), and proteases [3].

The beneficial effect of various enzymes in improving the growth and feed efficiency in poultry is well documented. For examples, a meta-analysis conducted by Swann and Romero investigated the effects of a mixture of xylanase, amylase, and protease on nutrient digestibility of broiler chickens. Their results showed that the enzyme combination increased the apparent digestibility of undigested crude protein, starch, and fat by 22.7, 88.9, and 33.4%, respectively [84]. McCormick et al. reported that the supplementation with 500 or 1500 FTU/kg of phytase in broiler diets significantly improved the growth performance, tibia ash, and apparent ileal digestibility and retention of phosphorus [85]. Guo et al. indicated that 5500 U/kg xylanase supplementation of wheat-based diets improved FCR in birds irrespective of C. perfringens infection and elevated apparent ileal digestibility of crude protein and mRNA expression of nutrient transporters in infected birds [86]. Nuseirat et al. recently showed that birds fed with the combination of xylanase (10XU/g feed) with probiotic Bacillus spp. (1x10⁵ CFU/g feed) showed the improvement of live performance, reducing environment microbial load, as well as improving energy utilization [87].

The supplementation of swine diets with exogenous enzymes to enhance performance is not a new concept, and research articles in this field date back to the 1950s. However, the response of pigs to supplementation with traditionally fermented enzymes is less consistent than has been observed with poultry. It is possible that exposure to the low pH in the stomach of the pig is either partially or totally denaturing the enzyme accounting for the lower magnitude of responses obtained when carbohydrases are fed to pigs compared with poultry [20]. Recently, several carbohydrases have been developed by genetic engineering, which have considerable potential for animal feed application. Diets supplemented with recombinant β-mannanase increased weight gain by 16.4% and feed efficiency by 17.7% in growing pigs, while pigs fed the diet with traditionally fermented β-mannanase improved BWG and FCR by 3.4 and 4.9%, respectively [88, 89].

3.1.5 Phytogenics

Phytogenic compounds have been widely recognized as potential alternatives to AGPs. Phytogenics, also referred as phytobiotics or botanicals, are plant-derived natural bioactive compounds used to enhance animal productivity and health [90]. A wide variety of plants and their extracts including herbs and spices (e.g., garlic, cumin, pepper, mint, cinnamon, turmeric, clove, alfalfa, thyme, sumac, aloe vera, mulberry leaf); essential oils (plant-extracted oils of the families Alliaceae (onion), Apiaceae (celery), Asteraceae (aster), Lamiaceae (oregano, thyme, lavender, peppermint, sage oils), Lauraceae (cinnamon oil), Liliaceae (garlic oil), Myrtaceae (tea tree oil), Poaceae (grass) and Rutaceae); and oleoresins (volatile and nonvolatile components responsible for the characteristic flavor and aroma) are often classified as the common phytogenic compounds [3, 15, 91]. In addition to these phytobiotics, essential oil nanoemulsion (NE) that is known as an isotropic mixture, a combination of oil and surfactant, which spontaneously forms fine emulsions of oil in water, is also considered as a type of potential phytogenics [92].

In recent years, the phytogenics used individually or as blends in feed have been investigated and shown positive effects on feed efficiency, antimicrobial, and immune stimulating in poultry and swine production. For examples, the supplementation with various levels and forms of Aloe vera [93, 94, 95, 96], garlic [97, 98, 99]; pepper [100, 101, 102, 103]; turmeric [104, 105, 106, 107] in feed improved the productivity of both broilers and laying hens, enhanced the immune responses, increased Lactobacillus counts, and reduced E. coli counts. Also, Guo et al. showed a significant increase in BWG and improvement in feed efficiency when broilers were given diets supplemented with a mixture of 14 herbs [108]. A similar study showed that a mixture of essential oils (EO) derived from caraway, basil, lemon, laurel, sage, oregano, thyme, and tea enhanced the growth of broilers [109]. Likewise, Eucalyptus and peppermint EO showed higher hemagglutiin-inhibition antibody titers against both avian influenza and Newcastle vaccines as compared with control [110]. However, the volatile bioactive components in the EO make it possess the antimicrobial activity, and also become a limiting factor in EO application. Nanoemulsions carrier systems can be a solution to tackle that problem. Nanoemulsion is increasingly being utilized for improving the bioavailability of certain types of volatile components, where most of them are lipophilic substances. Noori et al. found that nanoemulsion-based edible coating containing ginger EOs can help increase the life of breast fillets [111]. Similarly, Keykhosravy et al. also reported that edible chitosan-loaded nanoemulsions containing two essential oils (Zataria Multiflora Boiss and Bunium persicum Boiss) could play an effective role in the preservation of the microbial qualities of turkey meat [112].

A multitude of studies about effects of phytogenics on swine growth and health have been also investigated. For instances, a recent study reported that pigs fed diets with a phytogenic mixture (including garlic oil, cinnamic, aldehyde, thymol, carvacrol and eugenol) and/or encapsulated sodium butyrate increased ADG and FCR [113]. An earlier study also showed that piglets fed with herbal extracts (sage, lemon balm, nettle, and purple coneflower) grew faster than control animals and showed significantly higher final average body weights. The herbal extracts improved the structure of the ileal epithelium by considerably increasing the villus height. Better digestibility of nutrients could be due to higher villi in these animals [114]. Supplementing EO has been reported to improve the immune status of piglets after weaning, as indicated by an increase in lymphocyte proliferation rate, phagocytosis rate, as well as in IgG, IgA, IgM, C3, and C4 serum levels [115, 116, 117].

In addition, there is also limited information concerning the interaction between EO and feed ingredients. Jamroz et al. investigated the influence of diet type (corn vs. wheat and barley) on the ability of plant extracts (100 mg/kg containing 5% carvacrol, 3% cinnamaldehyde, and 2% of capsicum oleoresin) to modify morphological and histochemical characteristics of the stomach and jejunal walls in chickens [118]. Their results demonstrated significantly more jejunal wall villi in birds fed the maize diet with plant extracts. The incorporation of carvacrol, cinnamaldehyde, and capsicum oleoresin promoted positive and negative changes in digestive function, intestinal epithelium, microbial ecology, and fermentation in weaned pigs depending on the amount of protein included in the diet [119]. In general, phytogenics enhance the production of digestive secretions and nutrient absorption, reduce pathogenic stress in the gut, exert antioxidant properties, and reinforce the animal’s immune status, which help to explain the enhanced performance observed in swine and poultry [120].

3.1.6 Antibody therapy

Antibodies can cause agglutination of bacteria and viruses, thus lessening the number of infectious units, restrict mobility of the pathogen, and inhibit microbial metabolism and growth when antibodies bind to bacterial transporter proteins [121]. In poultry, maternal antibodies are transmitted to the offspring via the yolk of the eggs [122]. As a consequence, egg yolk was one of the sources of antibodies that has gained much interest as an inexpensive nonantibiotic alternative for prophylaxis and therapy of infectious diseases in an agricultural setting [123]. Several studies have successfully tested the ability of egg yolk immunoglobulins to control infectious diseases in chickens [3]. For examples, Lee et al. [124, 125], Xu et al. [126]; Juarez-Estrada et al. [123] demonstrated that the oral immunotherapy using egg yolk IgY against Eimeria sp. represents an effective and natural resource against severe E. tenella, E. acervulina, E. maxima infection favoring the gradual withdrawal of the anticoccidial drugs and antibiotics. Rahimi et al. investigated the effect of supplementation of Salmonella enteritidis-specific IgY on 3-day-old infected chicks and found lower fecal shedding and S. enteritidis concentration in the cecal content. They also observed a lower isolation of S. enteritidis from the liver, spleen, and ileum of birds [127]. Chalgoumi et al. also reported that antibodies simultaneously directed against S. enteritidis and Salmonella typhimurium can be efficiently produced in the same egg yolk of hens immunized with S. enteritidis— bacterial outer membrane proteins and S. typhimurium—bacterial outer membrane proteins in a half-dose mixture. This antibody mixture can be used as an additive in broiler chicken diets to fight both S. typhimurium and S. enteritidis, which are the predominant cause of salmonellosis in human often associated with poultry meat consumption [128]. In addition, Campylobacter jejuni is one of the most important causes of foodborne gastroenteritis. Chickens are considered a reservoir host of C. jejuni, and epidemiological studies have shown that contaminated chicken meat is a primary source of human infection. AI-Adwani et al. investigated the effect of IgY against the five C. jejuni colonization-associated proteins or CAPs (CadF, FlaA, MOMP, FlpA, and CmeC). They showed that α-CadF, α-MOMP, and α-CmeC IgY significantly reduced adherence of C. jejuni to the chicken hepatocellular carcinoma cells, suggesting that these α-C. jejuni CAP-specific IgY may be useful as a passive immunotherapeutic to reduce C. jejuni colonization in chickens [129].

Furthermore, it has been well documented that oral administration of IgY acts as potential AGPs for controlling diarrhea and exerting growth-promoting activity in pigs [130]. Diarrhea due to enterotoxigenic Escherichia coli (ETEC) is by far the most common enteric colibacillosis encountered in neonatal and post-weaned pigs. A research group of Jin et al. and Marquardt et al. investigated the effects of egg-yolk antibodies against ETEC K88 in in vitro piglet intestinal mucus and in neonatal and early-weaned piglets. The in vitro studies showed that anti-K88 antibodies from chicken egg-yolk when added to ETEC K88 prevented their binding to receptors in the mucus isolated from the intestine of piglets. Further, they also indicated that the neonatal and early-weaned piglets that received the egg-yolk antibodies were protected against ETEC infection [131, 132]. Porcine epidemic diarrhea virus (PEDV) is another important enteric viral pathogen that is responsible for neonatal piglet diarrhea. The studies of Weiping et al. and Cui et al. revealed that the survival rate was increased significantly in pigs treated with IgY compared with a control group suggesting that IgY can be an alternative method for conferring protection in piglets against PEDV [133, 134].

3.1.7 Antimicrobial peptides

The antimicrobial peptides (AMPs) are a class of short peptides widely found in nature, and they are an important part of the innate immune system of different organisms. They have inhibitory effects against bacteria, fungi, parasites, and viruses [135]. Since the emergence of antibiotic-resistant microorganisms, the AMPs have rapidly captured attention as potential drug candidates for replacement of antibiotics [135]. A recent review showed that there are more than 700 AMPs known to exist. And they are generally classified based on source, activity, structural characteristics, and amino acid-rich species [20]. Among AMPs, interest in bacteriocines has been widely increased due to their antibacterial properties [136], and a few hundred bacteriocines were currently described [137]. Numerous evidences showed the potentials of using bacteriocines to improve the animal growth performance and inhibit the pathogens growth. Ogunbanwo et al. investigated the potential therapeutic efficacy of bacteriocin and bacteriocin-producing Lactobacillus plantarum strain in an experimental E. coli infection of broiler chickens. They found that the significant reduction of clinical signs of colibacillosis, improvement in the growth rate of the studied birds, lower percentage of re-isolation of E. coli, and reduction of abnormally high globulin were exhibited in chickens infected with E. coli and are treated with bacteriocin or bacteriocin-producing L. plantarum [138]. Grilli et al. reported that pediocin A, a bacteriocin produced by Pediococcus pentosaceus, was highly active against C. perfringens in an in vitro assay [139]. They also showed that a partially purified fraction of pediocin A, alone or in association with the producer strain, significantly improved the growth performance of broiler chickens challenged with C. perfringens [139]. Wang et al. demonstrated the efficacy of bacteriocin (albusin B) as a potential alternative for feed antibiotics. In this study, the albusin B, which is produced by Ruminococcus albus 7, has been reported to improve broilers body weight gain, elevate mRNA expression of sGLT1, GLUT2, and PEPT1 in the jejunum, decrease Samonella load, and increase the fecal Lactobacillus counts [140]. Futhermore, nisin, one of the most commonly used bacteriocins for food preservation, has been also reported to enhance the growth performance and modulate GIT ecology of broilers [141].

Post-weaning diarrhea is responsible for major economic losses in the swine industry. ETEC is the major cause of this enteric disease in pigs, being responsible for approximately 50% of piglet mortality. Colicins, a class of bacteriocines, have been shown to be effective against ETEC strains and could be a potential alternative to antibiotics in swine production [142]. Culter et al. reported that dietary inclusion of colicin E1 was shown to decrease the incidence and severity of post-weaning diarrhea caused by F18-positive ETEC [142]. In addition to ETEC, Streptococcus suis is a major swine pathogen that has been associated with severe infections such as meningitis, arthritis, endocarditis, pneumonia, and septicemia, and major responsible for economic losses in the swine industry [143]. The nisin-producing strain L. lactis ATCC 11404 proved to be capable of inhibiting the growth of S. suis. And, all the S. suis isolates tested were susceptible to purified nisin, with the minimum inhibitory concentration ranging from 1.25 to 5 μg/mL [143]. Furthermore, Hu et al. have demonstrated that the Lactobacillus gasseri LA39 and Lactobacillus frumenti as potential microbes associated with diarrhea resistance in early-weaned piglets, and thus, microbiota-derived bacteriocin gassericin A targets host intestinal epithelium may also help prevent diarrhea suggesting that secretory gassericin A may also serve as a specific biomarker for diarrhea resistance in early-weaned piglets [144]. In general, bacteriocins not only represent alternatives to AGPs but are also considered as a promising therapy for preventing and controlling animal diseases.

3.1.8 Bacteriophage therapy

Bacteriophages are bacteria-specific viruses that have been utilized as therapy to against pathogens and are thus considered as alternatives to antibiotics in the age of multi-drug resistance. The bacteriophage is probably the most abundant biological entity on the earth with estimation of 10^31–32 phages [145]. Bacteriophages can be generally classified based on morphology, nucleic acid, phage life cycle, and bacterial target and site. Regarding the morphology, the tailed phages constitute the order Caudovirales, which is divided into families: Siphoviridae (61% of tailed phages), Myoviridae (25%), and Podoviridae (14%) families [146]. Further, phages are categorized into two types based on their life cycle, namely virulent and temperate. But temperate phages cannot be utilized as antimicrobial agents for therapeutic purposes because they may transfer genetic material from one bacterial cell to another. In contrast, virulent phages rapidly exterminate the bacteria, enabling them to be used as efficient antibacterial agents [147]. There has been an explosion of research and interest in the usage of bacteriophages in the poultry industry. Most of the phage-based products are targeted against the main foodborne pathogens, such as C. jejuni, Salmonella spp. (S. Enteritidis; S. Typhimurium), E. coli, Listeria monocytogenes, Staphylococcus aureus, and C. perfringens. A recent study demonstrated that a phage cocktail containing virulent Campylobacter phages was used by oral route to treat broiler chickens challenged with C. jejuni [148]. Bacteriophage predation of C. jejuni did not affect the microbiota but selectively reduced the numerous of C. jejuni. They have concluded that bacteriophage control to reduce C. jejuni levels in chickens could reduce human exposure and disease acquired through the consumption of contaminated poultry products [148]. The previous similar studies have been performed to investigate the effects of phages against Salmonella spp. infection in chicken. Bardina et al. and Hong et al. reported that a cocktail phage containing virulent Salmonella spp. significantly reduced the Salmonella cell numbers in chicken challenged with Samonella by oral administration [149, 150]. Consequently, in 2019, the first results were reported from the use of Salmonella phages at a commercial scale in the poultry production system [151]. In addition, E. coli-associated infections are widely distributed among poultry of all ages and categories. Huff et al. have demonstrated that aerosol spray of bacteriophages administered to 7-day-old chickens prior to the triple challenge with E. coli can prevent airsacculitis caused by E. coli [152]. Eid et al. indicated that phage therapy found to be an attractive option to prevent and control multidrug-resistant colibacillosis in broilers [153].

Furthermore, although phage therapy has been used successfully in swine since the early 1920s, it has only recently started to attract the attention of the research community as a tool for use against bacterial diseases in swine [147]. For instances, Kim et al.; Yan et al.; and Gebru et al. reported that dietary supplementation with anti-Salmonella phage improved dry matter, nitrogen, and energy digestibility for growing pigs, and thus improved the performance of pigs [154, 155, 156]. Albino et al. isolated a Salmonella phage belonging to the Podoviridae family, which significantly reduced Salmonella counts in an in vitro experiment [157]. Morita et al. investigated the characterization of a virulent bacteriophage, named PP01, specific for E. coli O157:H7 isolated from swine stool sample. The phage concentration in stool estimated was 4.2 × 10⁷ PFU/g of the E. coli O157:H7. The results indicated that phage PP01 might suppress its host E. coli O157:H7 in the GIT ecosystem [158].

3.1.9 Metal and clay minerals

The use of trace minerals as alternatives to antibiotics in animal production has been gaining increasing attention in the recent years. Copper (Cu), a crucial trace element involved in various physiology and biochemical processes, includes hemoglobin synthesis, wound healing, bone development, and more importantly serves as a cofactor for many metabolic enzymes [20, 159]. However, animals can only absorb a small fraction of Cu and the most is discharged into the environment. Hence, the use of Cu as a growth promoter is not only important to health but also to environmental issue. In recent years, many studies have reported Cu nanoparticles (Cu-NP) as a promising alternative to AGPs. The main purpose of using Cu-NP as feed additives in poultry and swine production is to improve the growth performance, and reduce the pathogen growth and excretion of Cu into the environment [159]. Zheng et al. indicated that broilers were fed with supplementation of 2 g/kg Cu-NP in diet and exhibited the regulation of the intestinal microflora, the growth of beneficial bacteria, and inhibition of harmful ones, enhanced nitrogen metabolism, and reduced ammonia emission [160]. Similarly, the addition of Cu-NP in pig diets also improved the digestibility of crude fat and energy, enhanced IgG γ-globulin and total globulin protein levels, and increased superoxide dismutase activity [161].

In addition to Cu, zinc is another essential trace mineral that plays an important role in cell proliferation, immune response, reproduction, gene regulation, and defense against damage, and also has been commonly used as a growth promoter in animal production [3]. Nguyen et al. reported that chickens fed with different levels of nanoscale metal components including iron, Cu, zinc oxide, and selenium exhibited improved growth (hen’s body weight at 38 aged weeks and egg weight ranged from 2.53 to 2.60 kg/hen and 50.86–51.55 g/egg, respectively), the more efficient absorption of feed minerals, consequently decreasing the risk of environmental pollution [162]. Thema et al. evaluated different combinations of a probiotic (B. licheniformis), an organic acid mixture (benzoic and fumaric acids), a protease enzyme, and chelated minerals (Cu, Zn, and Mn) as alternatives to zinc-bacitracin antibiotic. They concluded that the diets could replace zinc-bacitracin antibiotic in broiler diets as they promoted similar growth performance and carcass characteristics [163]. Furthermore, the use of zinc in the pig diets also has positive effect on the growth performance. Kociova et al. investigated the effect of two formulations of zinc phosphate-based nanoparticles (ZnA and ZnC NPs) on pig growth performance, intestinal microbiota, antioxidant status, and intestinal and liver morphology. They found that all piglet groups fed with ZnA exhibited significantly higher piglet weight gain. The substantial occurrence of E. coli virulence factors was found on day 5, mainly in fimbrillary antigen and thermostable toxins, except for piglets fed by ZnC. The antioxidant status was affected only by ZnA group of piglets. The positive changes in the liver and the intestinal morphology of piglets with NPs were also observed [164].

Moreover, clays are crystalline, hydrated aluminosilicate molecules composed of alkali and alkaline earth cations along with small amounts of various other elements. Clay minerals are also used in particular in animal nutrition due to their absorption and decontamination properties significantly contributing to the health of the animals [165, 166]. For instances, AI-Beitawi et al. [167] investigated the effect of three levels of nanoclay minerals (1, 1.5, and 2%) on growth performance, internal organs, and blood biochemistry of broiler chickens compared with vaccines and antibiotics. The results showed that 2% nanoclay minerals fed at the two intervals significantly improved the growth performance of broiler chickens. Blood biochemistry, high-density lipoprotein, which are known to be beneficial for humans, significantly increased by feeding 1.5% nanoclay minerals at the two ages compared with control groups and other treatments. A recent report on pig showed that supplementation of the diet with 3 g/kg of an aluminosilicate mineral product comprising 72.6% SiO₂, 8.18% Al₂O₃, 9.42% Fe₂O₃, 5.25% K₂O, and 1.41% Na₂O could increase weanling pig performance [168].

3.2 CRISPR-Cas9 approach: a gene editing tool to against antibiotic-resistant bacteria

The CRISRP-Cas9 is a promising gene editing tool for controlling the prevalence of antibiotic resistance genes in bacterial populations and eliminating pathogens with high precision [169]. Thus, in recent years, various studies investigated the potentials of CRISPR-Cas9 system as an alternative therapeutic to antibiotics [21, 170].

As above described, the bacteriophages, a tool for use against bacterial diseases, has been widely utilized in the veterinary medicine. However, due to the structural diversity of phages, traditional nanoparticle delivery strategies are not practical. Drug therapy delivery often includes using different nanoparticles to absorb treatment cargoes. But these methods are ineffective for large, non-symmetrical phage cargoes, and pore sizes are too small to absorb phages. A potential alternative approach has been recently investigated is development of phages encoded with CRISPR-Cas9 offering species specific delivery of novel antibacterials. Citorik et al. and Bikard et al. use phage encoded with CRISRP-Cas9 to target antibiotic resistance invirulent strains of E. coli and S. aureus, respectively [171, 172]. They observed that the addition of phage encoded with CRISPR-Cas9 resulted in rapid killing of specific bacteria. In brief, although CRISRP-Cas system is far from commercial use, this alternative approach is currently being explored and considered as revolutionary tool in the fight against antimicrobial resistance.

4. Conclusion

Alternatives to AGPs are essential tools for minimizing the antimicrobial resistance crisis, reducing antibiotic use, and increasing animal productivity. The promising alternative approaches could be probiotics, prebiotics, organic acids, enzymes, phytogenics, AMPs, hyperimmune antibody, bacteriophages therapy, and CRISRP-Cas9 system. And, it is also believed that there is no single alternative that can replace the current use of antibiotics. It, therefore, is anticipated that the controlled combination of alternatives and/or with advanced tools may help address the issue of antibiotic resistance significantly such as CRISRP-cas9 encoded phages, synbiotics, essential oils, mineral, recombination enzymes.