Avian Pathogenic Escherichia coli (APEC): An Overview of Virulence and Pathogenesis Factors, Zoonotic Potential, and Control Strategies

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in avian species, and recent reports have suggested APEC as a potential foodborne zoonotic pathogen. Herein, we discuss the virulence and pathogenesis factors of APEC, review the zoonotic potential, provide the current status of antibiotic resistance and progress in vaccine development, and summarize the alternative control measures being investigated. In addition to the known virulence factors, several other factors including quorum sensing system, secretion systems, two-component systems, transcriptional regulators, and genes associated with metabolism also contribute to APEC pathogenesis. The clear understanding of these factors will help in developing new effective treatments. The APEC isolates (particularly belonging to ST95 and ST131 or O1, O2, and O18) have genetic similarities and commonalities in virulence genes with human uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC) and abilities to cause urinary tract infections and meningitis in humans. Therefore, the zoonotic potential of APEC cannot be undervalued. APEC resistance to almost all classes of antibiotics, including carbapenems, has been already reported. There is a need for an effective APEC vaccine that can provide protection against diverse APEC serotypes. Alternative therapies, especially the virulence inhibitors, can provide a novel solution with less likelihood of developing resistance.


Introduction
Avian pathogenic Escherichia coli (APEC), an extra-intestinal pathogenic E. coli (ExPEC), causes diverse local and systemic infections in poultry, including chickens, turkeys, ducks, and many other avian species [1]. The most common infections caused by APEC in chickens are perihepatitis, airsacculitis, pericarditis, egg peritonitis, salphingitis, coligranuloma, omphalitis, cellulitis, and osteomyelitis/arthritis; these are commonly referred as avian colibacillosis [2]. APEC also causes swollen head syndrome in chickens and osteomyelitis complex in turkeys [2]. Colibacillosis is one of the leading causes of mortality (up to 20%) and morbidity in poultry and also results in decreased meat (2% decline in live weight, 2.7% deterioration in feed conversion ratio) and egg production (up to 20%), decreased hatching rates, and increased condemnation of carcasses (up to 43%) at slaughter [1,3,4]. Furthermore, APEC is responsible for high mortality (up to 53.5%) in young chickens [4]. Taken together, along with the treatment expenses, APEC costs the poultry industry hundreds of millions of dollars in economic losses worldwide [5]. In the United States (US), it has been estimated that economic losses to the broiler industry can be as high as $40 million annually only due to carcass condemnation [6].

Invasins
Invasins are a class of proteins associated with the entry of pathogens into host cells [2,31]. Invasins play a role in promoting entry during the initial stage of the infection [2,31]. Multiple genes encoding invasins, ibeA (also called ibe10), ibeB (invasion protein), tia (toxigenic invasion locus), and gimB (genetic island associated with neonatal meningitis) have been reported in APEC isolates [35,47,48]. In addition, invasins also contribute to APEC resistance to oxidative stress induced by macrophages, biofilm formation, colonization, and proliferation in the host [47,48]. IbeR, a regulator of ibeRAT operon, contributes to invasion, resistance to serum and environmental stresses, and the expression of virulence genes [49]. Similarly, ychO, a putative invasin gene, plays a role in motility, adhesion, invasion, biofilm formation, and the expression of membrane proteins and metabolism genes [50].

Secretion Systems
The secretion systems are cell-associated systems that are present on cell membranes of bacteria and function to secrete proteins into host cells, thereby causing damage to the host cells [11,12]. The secreted proteins promote the bacterial virulence either by directly intoxicating the host cells or by enhancing attachment to host cells, establishing replicative niche by scavenging resources and by competing with other microorganisms. Among the different bacterial secretion systems, two secretion systems (type III and VI) contribute to APEC pathogenesis [11,12]. The regulators (EtrA and YqeI) [12,78] and ATPase (EivC) [79] of the type III secretion system 2 (ETT2) play a role in motility, adhesion, intracellular survival, proliferation, colonization, resistance to phagocytosis and serum bactericidal activity, expression of fimbriae genes, and the downregulation of pro-inflammatory cytokine responses. Similarly, different components of type VI secretion system, DotU (organelle trafficking protein), IcmF (intracellular multiplication factor), Hcp (hemolysis co-regulation protein), CpxR, CpxA (envelope stress response system), ClpV (ATPase), and VrgG (secreted protein) mediate interbacterial competition, adhesion, invasion, intracellular survival, colonization, motility, biofilm formation, production of type 1 fimbriae, resistance to serum bactericidal activity, and modulation of intracellular host responses (IL-8, IL-1β) [11,40,[80][81][82][83][84][85].

Two-Component Systems
Two-component systems (TCS) are major signaling proteins in bacteria that enable bacteria to respond to changing environments by altering the expression of genes [86]. Different TCSs have been reported with a role in APEC pathogenesis [86][87][88][89]. A membraneassociated TCS, PhoPQ, plays a role in biofilm formation, motility, adhesion, invasion, intracellular survival, systemic dissemination, and the expression of virulence genes and genes associated with flagellar assembly, ABC transporters, quorum sensing, and bacterial chemotaxis [13,86,90]. Similarly, another membrane-associated TCS, BasSR, is involved in biofilm formation and APEC virulence and colonization in vivo [91]. KdpDE, a TCS regulating potassium transport, mediates the expression of flagella-related genes, flagellum formation, motility, and resistance to serum bactericidal activity [87]. Likewise, a TCS regulating nitrogen metabolism, RstAB, contributes to iron acquisition, acid resistance, intracellular survival, and colonization [88,92]. Another TCS, BarA-UvrY, plays a role in the adhesion, invasion, persistence, intracellular survival, resistance to serum bactericidal activity and oxidative stress, and regulation of exopolysaccharide production and expression of type 1 and P fimbriae [89].

Transcriptional Regulators
Multiple transcriptional regulators have shown a role in APEC pathogenesis [14,15,37,[93][94][95]. The AutA and AutR, two global transcriptional regulators, mediate the expression of K1 capsule and acid resistance systems, and change in adaptive lifestyle to facilitate infection [14]. FNR (fumarate and nitrate reduction), another global transcriptional regulator, facilitates the adhesion, invasion, expression of type 1 fimbriae and type VI secretion system, and resistance to oxidative stress [15]. McbR (MqsR-controlled colonic acid and biofilm regulator) plays a role in biofilm formation and stress response [94], whereas tyrR (a transcriptional regulator involved in the biosynthesis and transport of aromatic amino acids) promotes invasion, motility, and intracellular survival [37]. YjjQ (transcriptional regulator, LuxR family) contributes to flagellar motility [93], and RfaH, a transcriptional anti-terminator, contributes to invasion, intracellular survival, and resistance to serum bactericidal activity [95].
Overall, multiple virulence and pathogenesis factors of APEC are involved in causing colibacillosis in poultry. As a result of the involvement of multiple virulence and pathogenesis factors, there is a hindrance in developing therapeutics broadly effective against APEC infections. In-depth understanding of these factors as well as unraveling the new factors will help develop the effective therapeutics against colibacillosis in poultry. Furthermore, several of these factors have coordinated and overlapping functions, which necessitates a holistic strategy to formulate an ideal anti-APEC therapeutics. For instance, developing therapeutics targeting iron acquisition systems [112], QS system [113], bacterial metabolism [114], and secretion systems [115] can provide solutions to mitigate APEC infections in poultry in the future.

Zoonotic Potential
APEC belongs to the ExPEC subgroup of E. coli, similar to UPEC and NMEC [4]. Multiple studies have reported APEC as a potential foodborne zoonotic pathogen as well as a source or reservoir of extra-intestinal infections in humans [21,22,41,. This is in particular due to its shared genetic similarity with human ExPECs, the presence of common or human ExPEC-defining virulence genes, and the ability to cause UTI and meningitis in rodent models, similar to UPEC and NMEC.

Genetic Similarity and Commonality in Virulence Genes
Multiple studies have shown that APEC shares genetic similarities with human Ex-PECs (UPEC and NMEC) and avian-associated ColV plasmids (for instance, pAPEC-078-ColV, pAPEC-O2-ColV, pAPEC-O1, p1ColV5155) essential for poultry adaptation are present in human ExPEC isolates. The phylogenetic (single nucleotide polymorphisms; SNP) comparison of whole-genomes of 323 APEC and human ExPEC isolates belonging to sequence type ST95 revealed genetic overlap (no distinct clustering) between APEC and certain human ExPECs, indicating that certain ExPEC clones may have the potential to cause infection in both poultry and humans [116]. Especially, ColV plasmids specific to APEC were present in those human ExPEC isolates, and 10 virulence genes (iucC, iucD, iutA, cvaA, etsA, hlyF, ompT, cvaB, cvaC, and cvi) were common between them. Another study compared the whole genomes of 48 APEC and UPEC isolates belonging to ST131-H22 [21]. The high genetic similarity (no distinct lineage) was observed in SNP-based phylogenetic analysis between UPEC and APEC isolates together with the presence of ColV plasmids in UPEC isolates. The ColV/ColBM plasmids were also present in 34.5% of APEC and 18.9% of human ExPEC isolates when whole genomes of 551 mcr-1 positive APEC and human ExPEC isolates were analyzed [118]. Therefore, the presence of poultry-specific ColV plasmids in human ExPEC isolates might suggest a zoonotic transmission of APEC from poultry to humans.
Several other phylogenetic studies have also shown the similarity of APEC with human ExPEC isolates. The phylogenomic tree constructed based on whole genomes of 47 E. coli strains revealed that APEC isolates belonging to serogroups O1:K1 and O2:K1 share significant genetic similarities/overlap (same cluster) with human ExPEC O18:K1 strains [124]. This finding is further supported by several other studies [22,[125][126][127][128][129]136]. The ExPEC (APEC, UPEC, and NMEC) isolates belonging to ST95 and serogroups O1, O2, and O18 were clustered together when the phylogeny of ExPEC isolates was constructed based on the possession of different genes/traits or multilocus sequence typing (MLST), suggesting the potential transmission of certain ExPEC strains between poultry and humans [125,127]. Similarly, APEC isolates belonging to serogroup O18 were similar to NMEC strains when compared using MLST and pulsed-field gel electrophoresis (PFGE) [22,129]. In other studies, APEC isolates belonging to ST95 and serogroup O1 were also similar to UPEC and NMEC isolates when compared using MLST, PFGE, and whole genome analysis, suggesting the zoonotic potential of these isolates to humans with no host-specificity [126,128]. In several other studies performed using MLST and PFGE, APEC isolates belonging to sequence type other than ST95, such as ST359 [130], ST23 [131], ST10, ST117, ST746 [132], ST617, ST23 [135], or other serogroups such as O45 [133,134] were also similar to human ExPEC isolates. Therefore, the APEC isolates belonging to certain STs or serogroups could pose a significant zoonotic risk to humans.

Ability to Cause Disease with Similar Clinical Manifestations
Multiple studies have shown that APEC can cause UTI and meningitis similar to UPEC and NMEC, respectively [22,124,[137][138][139][140]. The E. coli isolates from chicken meat and shell eggs were lethal similar to UPEC in a mouse model of UTI, caused sepsis in a mouse sepsis model, and infected the cerebrospinal fluid (CSF) similar to NMEC in a neonatal rat meningitis model [137]. Furthermore, these isolates also possessed swimming (motility) and biofilm-forming ability in urine, and they adhere, invade, and survive intracellularly in human kidney and bladder cells, similar to UPEC. These isolates also possessed K1 capsule and ibeA, which are essential virulence factors for NMEC pathogenesis. Similar findings were also observed in other studies [22,41,124,[138][139][140]. The E. coli isolates from chicken feces or from colibacillosis lesions were also lethal, caused sepsis, and infected CSF in rodent model studies [41,138]. In another study, E. coli isolated from colibacillosis cases and belonging to phylogroup F were able to cause disease (sepsis, meningitis, and UTI) in animal models of human infections [41]. The APEC and ExPEC (UPEC and NMEC) isolates, belonging to ST95 when compared, both were equally able to adhere and invade kidney cells, form strong biofilm, and resist the bactericidal activity of serum [139], reinforcing the understanding that APEC isolates belonging to ST95 pose a potential zoonotic risk to humans. Other similar studies have found that APEC isolates, particularly belonging to serogroup O18, survive in human serum, and they bind and enter human macrophages and human cerebral microvascular endothelial cells, similar to NMEC [22,140]. These isolates also induce neuronal apoptosis in mice, suggesting that APEC O18 isolates utilize similar pathogenic mechanisms as NMEC to cause meningitis in mice. Furthermore, APEC isolates, belonging to serogroup O1:K1 and O2:K1, also cause sepsis and meningitis in rodent models, suggesting that APEC O1:K1 and O2:K1 can have zoonotic potential [124].
Conversely, UPEC isolates also induced colibacillosis in chickens [141,142]. The experimental infection of laying hens with UPEC isolate caused salphingitis similar to APEC [141]. UPEC also induced similar symptoms and lesions comparable to those caused by APEC in chickens [144]. In another study, UPEC isolates belonging to serogroups O4, O74, O1, and O75 were 100% lethal to chickens [142]. The NMEC isolates were also lethal to chick embryos and caused colisepticemia in chickens, similar to APEC [22].
Overall, the potential of APEC ST95 and ST131 strains to cause UTI and meningitis in humans through the consumption of contaminated poultry products signifies the zoonotic nature of APEC. Furthermore, the zoonotic potential of APEC isolates, especially belonging to serogroups O1, O2, and O18, cannot be underestimated; therefore, interventions appropriate to mitigate the transmission of APEC to humans should be undertaken to combat the food safety threat posed by APEC to humans. Future investigations should consider determining the link between APEC and potential zoonotic transmission to humans.

Control Strategies
The control of APEC infections in poultry relies on antibiotic medication and vaccination, other than managing the environmental stressors, applying the biosecurity measures, and vaccinating the chickens against the viral and immunosuppressive diseases [1,2,145]. Probiotics, bacteriophages, and different new alternatives (innate immune stimulants, virulence and growth inhibitors, and antimicrobial peptides) have been also evaluated [26][27][28][29][30] with a goal to develop effective preventative and therapeutic treatments to control colibacillosis in chickens. Potential checkpoints for controlling APEC infection in chickens are shown in Scheme 1.

Management and Biosecurity Measures
The management of environmental stressors such as ammonia and dust in poultry houses by maintaining good litter and air quality are some of the key factors in preventing APEC infections in poultry houses [1,2]. Proper ventilation as well as maintaining optimum temperature, humidity, and bird density help mitigate environmental stress in chickens [1,2]. Furthermore, the elimination of pre-disposing factors by vaccinating chickens against MG, IBV, NDV, and IBD reduces the incidence of APEC infections [1,2]. Good nutrition and birds with enhanced immune systems are also likely contributors to reducing the incidence of colibacillosis [1,2]. Moreover, the vertical transmission of APEC can be prevented at the breeding level or at the top of the production pyramid by different intervention measures such as developing breeds with increased resistance to APEC infections, cleaning and disinfection of hatching eggs, and minimizing the use of floor eggs [145]. The horizontal transmission of APEC can be limited by using all-in-all-out production systems, systematic culling of weak chicks at first week, and implementing effective sanitation programs [145]. The proper and efficient biosecurity measures together with feed and water (chlorination) decontamination and disinfection of poultry houses, feed mills, farm equipment, and premises are necessary to prevent APEC entry into farms [1,2]. The biosafety measures such as preventing access of vectors such as houseflies, wild birds, and rodents are also necessary to keep APEC out of poultry facilities [145].

Antibiotics
Antibiotics are commonly used to control APEC infections in poultry [23]. Many antibiotics belonging to different classes, such as tetracyclines (tetracycline, oxytetracycline, chlortetracycline), sulfonamides (sulfadimethoxine, trimethoprim, sulfadiazine, sulfamethazine, sulfaquinoxaline, ormethoprim), aminoglycosides (apramycin, gentamicin, neomycin, spectinomycin), penicillins (amoxicillin, ampicillin), cephalosporins (ceftiofur), quinolones (danofloxacin, sarafloxacin, enrofloxacin), polymyxins (colistin), chloramphenicols (florfenicol), macrolides (erythromycin), and lincosamides (lincomycin) have been used in poultry industry worldwide for the control of APEC infections [23]. However, APEC resistance to multiple antibiotics has been reported [32,34,35,51,53,54,[70][71][72][73]107,108,108,, which limits the use of these antibiotics and suggests a challenge ahead in using these antibiotics. Table 2 provides a summary of antibiotic resistance and resistance genes (mechanisms) reported worldwide in APEC isolates from chickens in the last five years (2015 to 2020). These data indicate APEC resistance to almost all classes of antibiotics, except carbapenems. Resistance to imipenem has been also recently reported [108,108,171]. The resistance is most commonly seen with ampicillin, tetracycline, trimethoprim, sulfamethoxazole, and streptomycin antibiotics. Importantly, a high level of APEC resistance to medically important antibiotics, such as β-lactams and colistin, have also been reported, which might pose a high risk to humans because of the transmission of antibiotic-resistant bacteria and genes through the food chain [173]. The strategies employed by the US and European Union (EU) to restrict the non-therapeutic use (for growth promotion) of antibiotics in food-animal production and to limit the therapeutic use (for treatment) of medically important antibiotics could aid in mitigating this risk [174]; however, benefits of such measures in curbing antibiotic resistance issues may take time to realize. The development of antibacterials solely for animal uses without cross-resistance potential or the identification of antibiotic alternatives as a replacement for antibiotics could aid in combating antibiotic resistance issues in the food-animal production.

Vaccines
Various vaccine candidates, mostly live-attenuated and recombinant vaccines, have been investigated to protect chickens against APEC infections [5,. Table 3 provides the summary of vaccines tested to date along with their main findings. In the past, inactivated vaccines were tested; however, recent studies have focused mostly evaluating live-attenuated and recombinant vaccines in chickens. The varying degrees of protection, ranging from none to partial to complete, have been achieved using these vaccines. Among the tested vaccines, multiple vaccines such as outer membrane vesicles (OMVs), bacterial ghost vaccines, recombinant iss, recombinant antigen (rAg) vaccine containing ExPEC antigens, Salmonella-delivered vaccines containing APEC antigens, ∆aroA, and ∆tonB/∆fur were able to reduce the mortality, lesions, and bacterial burden as well as stimulate the antibody (immunoglobulins; IgG and IgA) responses in chickens.  [190] Live-attenuated ∆tonB and ∆tonB/∆fur vaccine (E956) Layers (E956) Spray -Less lesions In airsacs, heart, and liver [184] Siderophore receptor and porin (SRP ® ) APEC vaccine for egg layer peritonitis Layers (O1, O2, and O78) I/V, I/VAG, I/T, I/P -Complete protection against mortality -Substantially reduce APEC colonization and lesions [206] Live-attenuated O78:K80 ∆aroA vaccine Chickens and turkeys (O78 and X) Spray or spray followed by oral booster -Significant reduction in mortality -homologous and heterologous protection [185]  Live-attenuated APEC O78:J29-∆crp vaccine (AESN1331) Layers (O78:J46) Spray, eye drop, and in ovo -Significant reduction in mortality -Less lesion scores in heart and liver [186] Live-attenuated S. typhimurium vaccine expressing O78 LPS and E. coli type 1 fimbriae Layers (χ7122, χ7252, and χ7096) Oral and spray -Lower lesion scores in airsacs -No heterologous protection [194] ∆galE, ∆purA, and ∆aroA (APECO78:EC99) vaccine Broilers (EC99 and EC317) Spray -High IgY level -Less lesion scores and APEC load -No heterologous protection [196] Live E. coli vaccine with rspL mutation (EC844) Broilers (EC317) Aerosol and oral -Lower APEC lesions with three doses [200] Recombinant -High IgA and IgG levels -Less lesions in heart, liver, and airsacs -Heterologous protection [188] Recombinant iss vaccine Layers (O2 and O78) S/C -High humoral response -Lower APEC lesions -Heterologous protection [195]   Despite multiple vaccine candidates showing proven efficacy in chickens in experimental studies, only two vaccines (live-attenuated APEC O78 ∆aroA Poulvac ® E. coli vaccine and inactivated Nobilis ® E. coli vaccine containing F11 fimbrial and FT flagellar antigens) are commercially available currently for use in chickens [5]. However, the major drawback of these vaccines is the lack of protection against heterogenous APEC infections [5]. The ideal APEC vaccine should be able to confer cross-protection against multiple APEC serotypes and be deliverable by mass-immunization methods, such as oral (feed or water) or spray routes [5]. The identification of common/conserved virulence and pathogenesis mechanisms employed by diverse APEC serotypes to cause disease in chickens would facilitate the development of new broad-spectrum vaccines. For instance, vaccine developed using outer membrane iron receptors required for iron acquisition (FyuA, Hma, IreA, IutA) protects against UPEC infections in a murine model [112]. In another study, a tetravalent conjugate vaccine developed using O antigens of predominant UPEC serotypes provides broad protection against UPEC infections [207]. Furthermore, the knowledge gained on new virulence and pathogenesis factors should be exploited to design potent vaccine candidates. For example, the type VI secretion system (vgrG) and quorum sensing system (luxS) can be the new vaccine targets because of their substantial involvement in APEC virulence and pathogenesis.

Probiotics
Different probiotics have been tested for their efficacy to prevent APEC infections in chickens [26,175,208,209]. The efficacy of Lactobacillus plantarum B1 was evaluated against E. coli (K88) infection by supplementing in the broilers feed (2 × 10 9 CFU/kg) [26]. Broilers fed with L. plantarum B1 showed significantly decreased cecal E. coli counts and increased growth performance, villus height to crypt depth ratio, ileal mucosal sIgA concentration, and cecal lactic acid bacteria counts. Similarly, the efficacy of L. plantarum 15-1 and fructooligosaccharides (FOS) combination was evaluated against APEC (O78) infection by supplementing in the broilers feed (1 × 10 8 CFU/kg) [208]. The decrease in mortality and serum diamine oxidase and increase in IgA and IgG concentrations was observed in broilers fed with probiotic and FOS mix. The effects of Enterococcus faecalis-1 was assessed in broiler chickens challenged with APEC (O78) by inoculating orally in drinking water for 3 days (1 × 10 8 CFU) from days 1 to 3 of growth [210]. E. faecalis-1 supplementation significantly improved the growth performance and immune response, reduced the mortality, and decreased the visceral organs invasion by APEC O78. Likewise, the efficacy of multi-strain commercial probiotic mix (Bacillus subtilis, Clostridium butyricum, and L. plantarum) was tested against APEC 078 infection by supplementing in the broilers feed [209]. There was significant decrease in mortality (13.6% to 0%) and APEC counts in liver and spleen and increase in growth performance and lactobacilli population in broilers fed with the probiotic mix. Another commercial probiotic mix (B. subtilis, L. acidophilus, Pediococcus acidilactici, Pediococcus pentosaceus, and Saccharomyces pastorianus) was also tested in combination with recombinant attenuated Salmonella vaccine (RASV) for protection against APEC (O78:K80) and Salmonella infection by supplementing in feed in layer chickens [175]. Chickens showed reduced signs of airsacculitis, perihepatitis, and pericarditis and lower APEC load in the blood. These studies suggest that different probiotics belonging to genus Lactobacillus, Bacillus, Clostridium, and Pediococcus show efficacy in preventing APEC infections as well as improve the growth performance, maintain the healthy intestinal microbiota, and enhance the intestinal mucosal immunity. Furthermore, there are multiple probiotic products (for example, Sav-A-Chick ® Probiotic Poultry Supplement, Probios ® , HealthyGutTM PROBI-OTICS, and SuperDFM-Poultry) commercially available for use in maintaining intestinal health and boosting immune status in poultry. These probiotics contain different beneficial microorganisms, such as B. subtilis, B. licheniformis, L. plantarum, L. casei, L. acidophilus, L. brevis, L. reuteri, Enterococcus faecium, E. thermophilus, P. acidilactici, P. pentosaceus, Bifidobacterium bifidum, B. animalis, Propionibacterium shermanii, and P. freudenerichii. Even though these probiotics are not indicated specifically for APEC, they can reduce the incidence of APEC infections in poultry farms due to their broad-spectrum effect against enteric pathogens. Furthermore, the identification of new probiotics with superior potential to protect against APEC infections is necessary, which can provide alternatives to antibiotics, thereby also mitigating the development of antibiotic resistance. For instance, the nextgeneration probiotics specific to APEC can be developed by investigating the microbiome of healthy and APEC infected chickens followed by the identification of beneficial bacteria crucial to resist APEC infection in chickens [211].

Bacteriophages
To date, multiple studies have been conducted to evaluate the preventative and therapeutic efficacy of phages against APEC infections in chickens [27,[212][213][214]. The efficacy of phage mixture (SPR02 and DAF6) was evaluated in APEC (O2) challenged chickens by spray and intramuscular administrations [214]. The phage treatment three days prior to APEC challenge significantly reduced (40% to 3%) the mortality of chickens. Similarly, phage treatment at 24 h and 48 h post-challenge also reduced the mortality rate (55% to below 20%). The efficacy of phage cocktail (phi F78E Myoviridiae, phi F258E Siphoviridae, and phi F61E Myoviridae) was tested in experimentally (O78) and naturally infected flocks refractive to antibiotic treatment by oral or spray administration [213]. The treatment with phage cocktail reduced mortality by 25% in experimentally infected chickens and decreased the flocks' mortality level to below 0.5% in flocks infected naturally with APEC. Similarly, the efficacy of another phage cocktail (TM1, TM2, TM3, and TM4) was evaluated in APEC challenged (O78:K80 and O2:K1) chickens by administering through intramuscular injection [212]. The phage cocktail treatment reduced the mortality (46.6% to 13.6%), APEC load in lung, and APEC lesions in lung, liver, and heart, and increased the body weight of chickens. The efficacy of phage-loaded chitosan nanoparticles (C-ΦKAZ14 NPs; Myxoviridae, T4-like coliphage) was also evaluated in APEC-challenged (O1:K1:H7) chickens by oral administration [27]. The C-ΦKAZ14 NP treatment decreased the mortality (58.33% to 16.7%), intestinal colonization of APEC (2.30 × 10 9 ± 0.02 to 0.79 × 10 3 ± 0.10 CFU/mL), and fecal shedding (2.35 × 10 9 ± 0.05 to 1.58 × 10 3 ± 0.06 CFU/mL). C-ΦKAZ14 NP treatment also increased the body weight of chickens as well as ameliorated the clinical signs and symptoms. These studies suggest that the phage therapy can be a valuable approach to control APEC infections in chickens. However, no treatment involving phages has yet advanced into field applications. This is partly due to challenges in large-scale production and controversies associated with approval for use in poultry production [215]. If proven safe and effective in field settings, phages can serve as a valuable alternative to antibiotics in poultry production.

Innate Immune Stimulants
Innate immune stimulants can activate the innate immune responses by acting as pathogen-associated molecular patterns (PAMPs) and binding to pattern recognition receptors (PRRs); thereby, preventing the host from infection [217]. The synthetic oligodeoxynucleotides containing unmethylated cytosine-phosphodiester-guanine motifs (CpG-ODN) was tested to protect neonatal chickens against APEC (O2) infection by administering through the intrapulmonary route using compressor nebulizer in an acrylic chamber [217]. Higher survival, better clinical conditions, and lower bacterial load were observed in chickens that received CpG-ODN. Furthermore, CpG-ODN also induced systemic antibacterial immune responses, including upregulation of expression of pro-inflammatory (IL-1β, LPS-induced tumor necrosis factor, IL-18) and anti-inflammatory (IL-10, IL-4) cytokine genes, and enrichment and maturation (higher CD40 and MHCII expression) of monocytes/macrophages and CD4 + and CD8 + T-cell subsets. Similarly, the prophylactic potential of three in ovo-administered innate immune stimulants and immune adjuvants (CpG-ODN, polyinosinic:polycytidic acid, and polyphosphazene) was evaluated to protect young chickens from yolk sac infection caused by APEC (O2) [28]. CpG-ODN increased the survival (>80%) of young chickens. Overall, these studies demonstrate that CpG-ODN can induce protective immunity against APEC-induced yolk sac infection in neonatal chickens. The evaluation in adult chickens to prevent APEC-associated infections is warranted to develop innate immune stimulants as new non-antibiotic measures for colibacillosis prevention.

APEC Virulence and Growth Inhibitors
Virulence inhibitors disarm/attenuate pathogens by inhibiting virulence mechanisms, such as the QS system, unlike the antibiotics that inhibit the bacterial growth [223]. Therefore, virulence inhibitors can overcome the limitations of current antibiotics, viz. resistance and killing of commensal bacteria, and making pathogens susceptible to natural host defenses; thus, they are superior to conventional antibiotics [223]. On the other hand, growth inhibitors possessing novel scaffolds with newer antibacterial targets with less likelihood for resistance development such as those targeting bacterial membrane can be promising new antibacterial agents [224].
The protective effect of baicalin (medicinal ingredient isolated from dry roots of Scutellaria baicalensis), an APEC QS inhibitor, was evaluated against APEC-induced acute lung injury (O78) in chickens [218]. The pre-treatment of baicalin (200 mg/kg) significantly reduced the mortality, lesion in lung, lung wet/dry ratio, myeloperoxidase activity, and levels of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) in the lung. In another study, the effect of rutin (flavonoid extracted from plants), an APEC QS inhibitor, was investigated on AI-2 secretion (APEC O78), biofilm formation, and the expression of virulence genes [29]. Rutin significantly reduced the AI-2 secretion, biofilm formation, and expression of virulence genes together with a decrease in adhesion and damage to chicken type II pneumocytes. Multiple APEC small molecule QS inhibitors, mostly piperazines, effective in reducing AI-2 secretion, biofilm formation, motility, the expression of virulence genes, and intracellular survival in macrophage and epithelial cells were identified through screening of the small molecule (SM) library [222]. The treatment with these QS inhibitors also increased the survival of wax moth (Galleria mellonella) larvae and decreased the intra-larval APEC load. Among the identified QS inhibitors, two QS inhibitors [QSI-5 (C-5) and QSI-10 (C-10)] [222] significantly reduced the APEC-induced mortality, lesions, and APEC load in internal organs of chickens (data not published). The effect of andrographolide (Andrographis paniculata) in reducing cell damage caused by APEC O78 was investigated in chicken type II pneumocytes [220]. Andrographolide significantly reduced AI-2 secretion, expression of virulence genes, the release of lactate dehydrogenase (LDH), F-actin cytoskeleton polymerization, and adhesion to chicken type II pneumocytes.
Multiple APEC small molecule growth inhibitors possessing pyrrolidinyl, imidazole, piperidine, quinoline, and nitrophenyl scaffolds were identified in our study through screening of the SM library [221,225]. These inhibitors were bactericidal to APEC, at very low concentrations, affected APEC membrane integrity, and decreased the intracellular survival of APEC in macrophage and epithelial cells. The treatment with these inhibitors also increased the survival of wax moth larvae and decreased the intra-larval APEC load. Among the identified growth inhibitors, two growth inhibitors [GI-7 (SM7) and GI-10 (SM10)] [221] significantly reduced the APEC-induced mortality, lesions, and APEC load in internal organs of chickens (data not published).
Altogether, various virulence and growth inhibitors have shown the potential to be developed as novel anti-APEC therapeutics. However, further efforts are needed to advance these inhibitors into field applications.

Antimicrobial Peptides
Antimicrobial peptides (AMPs), regarded as new category of therapeutic agents, are short and generally positive charged peptides [226]. AMPs have fast and selective antimicrobial action, even against antibiotic-resistant bacteria, with low propensity for resistance development, which makes them ideal candidates for antibacterial development [226]. The protective effect of prophylactic in ovo treatment of D analog of chicken cathelicidin-2 (D-CATH-2; host defense peptide) was evaluated against APEC (O78:K80) infection in chickens [219]. The treatment of D-CATH-2 reduced the mortality (by 63%) and bacterial load (>90%) along with the increment of IgM level and peripheral blood lymphocytes and heterophils. In another study, the efficacy of orally administered surfactinamoxicillin combination was evaluated against APEC (O78) infection in chickens [216]. Surfactin (lipopeptide) combination (0.01 mg/g) enhanced the efficacy (significant decrease in mortality, APEC load, and APEC lesions compared to amoxicillin treatment alone) of otherwise ineffective amoxicillin. The treatment also resulted in upregulation of expression of pro-inflammatory and anti-inflammatory cytokine genes (IL-1β, TNF-α, IL-10 and IL-13). In other studies, peptides (A3, P5, cecropin A-D-Asn, cLF36) have shown efficacy in decreasing the E. coli load in the chicken gut [30,227]. These studies demonstrate that antimicrobial peptides can be developed either as an alternative to antibiotics or as an adjuvant to antibiotics to enhance the efficacy of antibiotics.

Conclusions and Future Perspectives
APEC is a most common bacterial pathogen of poultry that causes significant economic losses to the poultry industry worldwide. The effective control of APEC is beneficial to both animal and human health. Multiple virulence and pathogenesis factors of APEC are involved in a coordinated way to cause systemic infections in poultry; therefore, a holistic approach encompassing all factors such as iron acquisition systems, QS system, bacterial metabolism, and secretion systems is necessary to formulate effective anti-APEC therapeutics in the future. Further investigation is necessary to provide concrete evidence for zoonotic transmission of APEC to humans. APEC isolates, particularly belonging to ST95 and ST131 or O1, O2, and O18 serogroups might serve as a source of human extraintestinal infections. As a result of the significant antibiotic resistance issues and high risk of transmission of antibiotic-resistant bacteria and genes to humans, the development of antibacterials solely for animal uses without cross-resistance to current antibiotics might provide a solution for the future. Furthermore, there is a need for an ideal APEC vaccine that can provide cross-protection against multiple APEC serotypes. Knowledge gained on virulence and pathogenesis mechanisms of APEC should be exploited to identify the new vaccine candidates. Lastly, alternative therapies should also be considered for further