Live Bacterial Prophylactics in Modern Poultry

Commercial poultry farms frequently use live bacterial prophylactics like vaccines and probiotics to prevent bacterial infections. Due to the emergence of antibiotic-resistant bacteria in poultry animals, a closer examination into the health benefits and limitations of commercial, live prophylactics as an alternative to antibiotics is urgently needed. In this review, we summarize the peer-reviewed literature of several commercial live bacterial vaccines and probiotics. Per our estimation, there is a paucity of peer-reviewed published research regarding these products, making repeatability, product-comparison, and understanding biological mechanisms difficult. Furthermore, we briefly-outline significant issues such as probiotic-label accuracy, lack of commercially available live bacterial vaccines for major poultry-related bacteria such as Campylobacter and Clostridium perfringens, as well research gaps (i.e., probiotic-mediated vaccine adjuvancy, gut-brain-microbiota axis). Increased emphasis on these areas would open several avenues for research, ranging from improving protection against bacterial pathogens to using these prophylactics to modulate animal behavior.


INTRODUCTION
Poultry animals like layers and broilers are some of the most critical food animals, with 90 billion tons of chickens meat being produced globally per year (1) and 290 eggs consumed per capita in the United States (2). Over the years, poultry have been domesticated to maximize particular functions like meat and egg production. Although selecting for greater weight gain and egg-laying rates has improved poultry productivity, specific selection for bacterial diseases resistance has not been pursued as diligently. This is problematic, as poultry animals are becoming increasingly at-risk for bacterial infections given the push for cage-free rearing [reviewed by (3)] and serve as major reservoirs for foodborne pathogens like Salmonella and Campylobacter, contaminating their products [(4); reviewed in (5)]. Furthermore, the emergence of antimicrobial-resistant (AMR) bacterial pathogens threaten poultry animals and humans health alike (6). Specifically, avian pathogenic Escherichia coli (APEC), Pasteurella multocida, and Mycoplasma gallisepticum are causal agents of disease and mortality in poultry animals, which have the potential to harbor AMR genes [ (7)(8)(9); reviewed in (10)]. Additionally, chickens are common carriers of bacteria like Salmonella and Campylobacter, which reside as commensals in their gastrointestinal tract [reviewed in (11,12)]. However, these bacteria are frequent contaminators of poultry products and cause gastrointestinal disease in human consumers [reviewed in (13)(14)(15)]. Even worse, these microbes can horizontally-exchange AMR genes with commensals or other pathogens (16)(17)(18). This has created a dangerous situation in which bacterial pathogens (chicken and human alike) may become highly-difficult to treat with conventional antibiotics. Thus, costeffective additives that can boost resistance to pathogenic and AMR bacteria are needed to further optimize both poultry health and productivity.
Among the strategies currently used to promote productivity in animal agriculture includes use of live microorganisms. This includes live bacterial vaccines, which are attenuated bacteria typically used to immunize animals against particular pathogens [reviewed in (19)], and probiotics, which are live, non-attenuated microbes that confer health benefits to the animal host (20). Probiotics are typically delivered via feed, although spray and intraocular administration are commonly-used to deliver live bacterial vaccines. Additionally, both live bacterial vaccines and probiotics can be cultured in vitro, which drastically lowers production costs [reviewed in (21); reviewed in (22)]. In this review, we will outline live bacterial vaccines and probiotics commercially-available in poultry, describing the peer-reviewed studies using these commercial products in poultry animals. Additionally, we discuss probiotic labels and reliability-concerns. Lastly, this review will discuss the potential for novel live vaccines, synergism between live prophylactics, and a possible role for live prophylactics in less-studied biological mechanisms such as behavior.

LIVE BACTERIAL VACCINES General
The earliest recorded live bacterial vaccination was in 1884, where Spanish clinicians utilized a weak Vibrio cholerae isolate to combat cholera outbreaks (23). Techniques for purposefullyattenuating bacterial strains while maintaining immunogenicity have been improved, using targeted modifications at the genetic level (24). Concerns over virulence-reversion by live bacterial vaccines have driven researchers to develop antigenbased vaccines incapable of sustaining disease. However, the successful development of antigen vaccines with long-term efficacy is relatively rare, mainly due to evolutionary adaptations by pathogens (i.e., antigenic loss/drift, serotype diversity) and design, as antigens have much-fewer epitopes compared to live bacteria vaccines, limiting protection against multiple, antigenically-diverse strains of a certain pathogen (25). Thus, live bacterial vaccines provide a lucrative alternative that can circumvent many issues with vaccination in poultry.
Like their wild-type counterparts, live bacterial vaccines can be easily cultured in vitro with low input costs, providing an inexpensive means of manufacturing large quantities of vaccine vs. the protein extraction steps required for antigenbased vaccines (21). Therefore, these vaccines can simultaneously prevent disease caused by their wild-type parent bacterium as well as additional pathogens (bacterial, viral, etc.) because cross-reactivity or via genetic insertions of genes encoding foreign antigen (24), creating an avenue for broad protection unachievable by many prophylactics currently available. In this review, we summarize characteristics and peer-reviewed findings for commercial live Salmonella enterica, Escherichia coli, Mycoplasma gallisepticum, and Pasteurella multocida vaccines available for poultry in Table 1.

Salmonella
Although Salmonella enterica induces inflammatory in the chicken gut at an early age (46-48), this bacterium can persist by restructuring the intestinal environment to promote immunological tolerance, allowing for asymptomaticallyshedding via feces from poultry animals [reviewed in (11)], resulting in potential contamination of meat and egg products. Human consumption of these contaminated poultry products is one of the major routes of salmonellosis incidence in the United States (49, 50). Live Salmonella vaccines are typically delivered orally via spray or drinking water to reduce Salmonella load in poultry. To improve food safety, live Salmonella vaccines are augmented with genetic deficiencies to limit intestinal replication while maintaining high levels of immunogenicity (24), although serotype and genetic attenuations are important drivers of vaccine efficacy (51, 52). Furthermore, these vaccines can be readily-modified to carry exogenous antigens (53, 54), enabling protection against additional pathogens.
Megan R Vac-1 is a cya crp S. Typhimurium vaccine [parent strain 3761 or UK-1 (55)], genetically-attenuated to knockout adenylate cyclase ( cya) and cAMP receptor protein ( crp). These mutations reduce pathogenicity and persistence of this live vaccine in the intestine while maintaining high immunogenicity, as demonstrated by the decrease of a challenge Salmonella invasion and intestinal colonization in vaccinated layer pullets (26). However, the protection of this vaccine against Salmonella appears to be inconsistent. A previous study testing protection against a wild-type S. Typhimurium strain in broiler chicks found the Megan R Vac-1 only reduced challenge Salmonella load in one of the two challenge experiments, although the failure in the first experiment may have been related to in ovo antibiotic administration (27). Furthermore, the vaccine strain was frequently-recovered from internal organs and ceca of vaccinated birds (27), although sampled animals were only 1-week-old and thus are not representative of broilers at final slaughter. In support of this, Dórea and colleagues determined that Megan R Vac-1 significantly-reduced detection of Salmonella in commercial broiler carcasses, minimizing carcass condemnation (28).
Poulvac R ST (Zoetis) is another metabolically-attenuated S. Typhimurium strain with serC (phosphoserine aminotransferase) and aroA (3-phosphoshikimate 1carboxyvinyltransferase) deletions. Despite these deletions, Poulvac R ST is still immunogenic, inducing antilipopolysaccharide IgA and IgY responses in intestinal washes at day 13 (29) despite a reduction in ileal macrophages and CD4 + T cells (30). Furthermore, vaccinated broilers had reduced S. Heidelberg loads in the ceca when challenged at 21 days old (31). This response may have been facilitated by recruitment of intestinal CD8 + T cells (30), which have been previously demonstrated to improve Salmonella clearance in chickens (56). However, this vaccine was unable to reduce Salmonella Heidelberg load in the ceca when challenged at 3 days (31). This may be due to serovar-specific, as Bailey and colleagues found that Poulvac R ST alone did reduce challenge Salmonella Typhimurium and Enteritidis invasion of internal organs and ceca colonization in 3 and 13-day-old chicks (29). Unlike the previously-described Salmonella vaccines, Gallivac R SE (Merial Select) is a S. Enteritidis strain ( ade his) developed via chemical mutagenesis. Similar to the other vaccines, Gallivac R SE can provide protection against non-Enteritidis serovars, as orally-delivered Gallivac R SE reduced S. Typhimurium burden in the liver and ceca up to week 71 in layer hens vs. unvaccinated hens (32). Although live Salmonella vaccines are normally given orally, intraocular administration of Gallivac R SE increased IFN , IL-8, and iNOs production by splenic cells (33), suggesting that this vaccine is capable of inducing robust immune responses, which extend from mucosal barriers. Unfortunately, to the authors' knowledge, these are the only two peer-reviewed studies which investigated the immunological potential of Gallivac R SE in vivo.

Escherichia coli
One of the major drivers of mortality and carcass condemnation in poultry, APEC are a major problem in commercial production (57). In addition, APEC are characterized by the possession of large virulence plasmids that often carry numerous resistances to antibiotics and heavy metals [reviewed in (58-60)]. These plasmids can be horizontally-transferred to other gut commensals as well bacteria like Salmonella (61), making the reduction of APEC in poultry a major priority. Given their antigenic variability (62), vaccines with broad protection have proved problematic. Notably, APEC colonize the gastrointestinal tract as commensals (63, 64) and only cause colibacillosis when they translocate the lung epithelium upon fecal aerosolization (65,66). Thus, orally-delivered live vaccines are a feasible strategy to reduce abundances of these microbes in the gut while also inducing systemic immunity for extraintestinal resistance.
As of this review, Poulvac R E. coli (Zoetis) is the only live E. coli vaccine for poultry on the market. Poulvac R E. coli has a O78 serotype and, similar to Poulvac R ST, is a aroA mutant. When implemented in broilers, this vaccine increased the number of healthy carcasses and reduced collibacillosis of a O78 APEC field isolate compared to non-vaccinated controls (67). Similarly, Poulvac R E. coli decreased bacterial load of an O78 APEC in internal organs compared to non-vaccinated controls, possibly related to improvements in E.coli O78 antigen-specific IgY serum levels and splenocyte proliferation (34). However, this protection appears to be serotype-specific, as Poulvac R E. coli did not confer any protection against challenge with an O1 APEC serotype (35). Furthermore, route appears to be a major determinant of efficacy, as Poulvac R E. coli administered to broilers via drinking water did not confer any protection to an O78 APEC, whereas coarse spray-administration did (35).

Mycoplasma gallisepticum
The etiological agent of chronic respiratory disease and infectious sinusitis in poultry animals [reviewed in (68); reviewed in (69)], M. gallisepticum is a major cause of carcass condemnation, reductions in egg-laying efficiency and weight gain, and mortality in commercial poultry (70)(71)(72)(73). Given its antigen variability, details regarding its entire pathogenesis are unclear (74). Initially, M. gallisepticum binds to sialic acid residues on lung epithelial cells (75) and can cause damage high inflammatory damage locally (76) or in deeper lymphoid tissues like the bursa of Fabricius (77). Although birds at all ages are susceptible to this bacterium, immunocompromised birds are especially at-risk for infection (78). Currently, live vaccines are typically used to prevent M. gallisepticum infection in poultry.
MG TS-11 (Merial Select) is a live attenuated strain of M. gallisepticum that is delivered via intraocular route (i.e., eyedrop). Its complete genome sequence is available to the public (79). This vaccine strain can prevent development of clinical airsacculitis, peribronchitis, and interstitial pneumonia via R-strain M. gallisepticum challenge without reducing egglaying productivity (36, 37). More recently, research groups have sought to improve the efficacy of the TS-11 vaccine. Muneta and colleagues found that a recombinant TS-11 expressing IFN increased cellular immunity via increased splenocyte-IFN production and a non-edematous infiltration of heterophils into the trachea mucosa (36). Furthermore, TS-304, a TS-11 derivative that expresses the cytadherence molecule GapA, was shown to be more efficacious than TS-11 at a lower dose (80) likely related to its ability to more-effectively improve tracheal barrier function (76).
MYCOVAC-L R (Merck) is an attenuated 6/85 strain of M. gallisepticum. Similar to TS-11, its complete genome sequence is readily-available (38). Typically delivered via spray, rehydration of the vaccine via distilled water (standard practice for many bacterial vaccines) results in much lower MYCOVAC-L R viability vs. resuspension in PBS (38). In addition, although vaccination dose at the manufacturer's recommendation confers protective immunity against virulent M. gallisepticum, egg production may be negatively-impacted. However, hens previously vaccinated with fifteen times the recommended dose did not exhibit any deficiencies in egglaying efficiency and produced more antibodies (39), suggesting a greater inoculum concentration is needed to negate certain side effects of MYCOVAC-L R .
Poulvac R MycoF (Zoetis) is an F strain of M. gallisepticum typically administered via spray. Using spray, Evans and colleagues showed MycoF-vaccinated animals did not exhibit M. gallisepticum-induced airsacculitis compared to control animals (40). Similar to MYCOVAC-L R , resuspension medium prior to MycoF immunization had a major impact on viability and antibody production immediately post-vaccination (81). When given via intraocular route, MycoF demonstrated protection against spread of M. gallisepticum in a co-mingled poultry system (82), suggesting that different vaccination routes may deliver similar success. However, when delivered in the same study via eyedrop, nares, or orally, intraocular MycoF vaccination induced the greatest antibody response (41), although this study did not investigate differences in M. gallisepticum resistance in vivo.
Another F strain vaccine, AviPro R MG-F (Elanco) was similarly able prevent airsacculitis via M. gallisepticum challenge (40). Although recommended delivery is in drinking water, Evans and colleagues found that when delivered via spray, MG-F delivered similar protection against M. gallisepticum infection as MycoF. Additionally, MG-F induced less antibody production vs. MycoF at one and ten-times recommended dose (42). However, MG-F induced superior immune responses compared to TS-11 and MYCOVAC-L R live vaccines (43, 44), suggesting that these M. gallisepticum vaccines induce immune responses in a vaccine strain-specific manner.

Pasteurella multocida
In the 1880s, Louis Pasteur developed one of the earliest live bacterial vaccines by isolating avian P. multocida, the etiological agent of fowl cholera, and using old cultures for immunization (83). Although a commensal member of the oropharyngeal microbiota, P. multocida can become an opportunistic pathogen in the respiratory tract (84). If able to bypass the lung epithelium, it can induce a highly-lethal septicemia (i.e., fowl cholera), causing major economic losses in poultry production (85,86), though turkeys are more-affected (85). Thus, wing-web immunization of live P. multocida vaccines, superior to bacterin-based vaccines for this pathogen (87), is the most common method of prophylaxes against this pathogen. Although the exact mechanisms for protection are somewhat unclear, these live vaccines can induce broad protection independent of serotype and lipopolysaccharide composition (88). However, to the author's knowledge, very little peerreviewed research has been performed using these Pasteurella live vaccines.
M-NINEVAX R -C (Merck) is an M-9 vaccine strain used in vaccinating commercial turkey flock against P. multocida and in combination with other live vaccines (89). Of the few studies using this vaccine, Sharaf and colleagues found this vaccine induced a potent antibody response against P. multocida (45). Similarly, PM-ONEVAX R -C, a PM-1 strain, induces protection against P. multocida challenge in vivo, accompanied with a high antibody titer (90). Unfortunately, no peer-reviewed studies on these live vaccines have been performed in the last two decades.

PROBIOTICS General
Probiotics are live microorganisms including bacteria (i.e., Lactobacillus acidophilus) and yeast (i.e., Saccharomyces cerevisiae) that are commonly supplemented in poultry feed to improve animal well-being through a variety of mechanisms. Probiotics have a variety of functions in host, which are mainly triggered by their outer membrane composition and metabolic outputs. In this section, we will discuss major classes of probiotics used in poultry and their general functions. Furthermore, we will summarize the findings of peer-reviewed studies using commercial probiotic products, organized by probiotics composed of a single class or mixture of classes ( Table 2). This review will be limited to the effects of these commercial products on host immune function, productivity measures, and bacterial resistance (specifically, intestinal colonizers like Salmonella, Campylobacter, E. coli, and Clostridium perfringens). Given the limited focus on mechanisms with these commercial probiotics  (118) in poultry, this review will outline observed outcomes in-general in peer-reviewed studies.

Lactic Acid Bacteria
Lactobacillus, Enterococcus, and Pediococcus are gut commensals and examples of lactic acid (i.e., lactate in the ionized form)producing bacteria (LAB), which protect against pathogens by several mechanisms. LAB are frequently used by poultry producers in part due their ability to produce several digestive enzymes (amylases, chitinases, lipases, phytases, and proteases), which greatly enhance the digestive process and improve feed conversion [reviewed in (120)]. Lactate is the major product of sugar metabolism across all LAB (121). Lactate can inhibit pathogenic bacterial growth by lowering the pH of the intestinal environment (122) or directly by disturbing normal bacterial metabolism (123). Select LAB also produce inhibitory compounds like bacteriocins, which are bactericidal compounds that target specific microorganisms (124). LAB can also directly stimulate immune cells via secretory factors (125) and toll-like receptor stimulation (125,126). Given this wide array of functions, LAB are common components in several commercial probiotics used in poultry agriculture. Some examples of commercial LAB products for poultry animals are discussed below. FloraMax R -B11 is a probiotic supplement composed of Lactobacillus salivarius and Pediococcus parvulus. Upon oral challenge with Salmonella Enteritidis, broilers fed FloraMax R -B11 showed reduced colonization of Salmonella Enteritidis, improved gut barrier function, and reduced percentages of heterophils, lymphocytes, eosinophils, and basophils of peripheral blood compared to control broilers (92). Given the role of immune inflammation in clearing intestinal Salmonella (127) and observed-reduction of circulatory immune cells, this suggests this product may have directly-reduced Salmonella load in the intestine. This mechanism of direct competition is supported in which each probiotic bacterium in FloraMax R -B11 directly-reduced Salmonella Enteritidis, E. coli, and Campylobacter jejuni growth in vitro (93). Although Prado-Rebolledo and colleagues did not investigate phenotypic changes in these immune cells, FloraMax R -B11 reduces intestinal gene expression associated with the NFκB complex and aldose reductase (91), suggesting this probiotic also reduces expression of inflammatory genes. In combination with the perinatal supplement EarlyBird (Pacific Vet Group USA Inc.), FloraMax R -B11 was shown to improve gut morphology and significantly decrease Salmonella recovery, incidence, and horizontal transmission to broiler chicks (94). Lastly, broilers supplemented with FloraMax R -B11 showed significant body weight gain, lower total Clostridium perfringens (the causal agent of necrotic enteritis), and lower necrotic enteritis-induced mortality when compared to control broilers after C. perfringens challenge (95).
Cylactin R is composed of a single LAB, Enterococcus faecium NCIMB 1045 (128). Implementation of Cylactin R to the diets of broilers has shown to have positive effects on average body weight, greatly-decreased counts of Clostridium spp. and E. coli in intestinal tract and excreta compared to controls, and had improved lactate production as well as short-chain and branched-chain fatty acids (96). However, Cylactin R alone did not reduce Salmonella Enteritidis load in the layer intestine (97). Although tested in a non-avian model, administration of Cylactin R in the diet of piglets showed significantly reduced mucus-adherent extraintestinal pathogenic strains of E. coli (129), suggesting that this probiotic could have direct effects on APEC found in the chicken intestine.

Bacillus
Similarly to LAB, Bacillus species secrete digestive enzymes that improve feed conversion and competitive exclusion, which limit the ability of pathogens to invade the host (130)(131)(132). However, B. subtilis specifically limits pathogen colonization by production and secretion of lipopeptides and other antimicrobial compounds, as 4-5% of a B. subtilis genome is devoted to the production of antimicrobials [reviewed in (133)]. In contrast to LAB, B. subtilis can form endospores (134), improving their survival in the harsh conditions of the intestinal tract and food preparation processes better than other probiotics (135,136). B. subtilis has also been shown to alter the morphology of the intestinal tract via elevated villi height and increased villi height-to-crypt depths (137), increasing the surface area for nutrient absorption. Notably, the host immune response toward B. subtilis is driven based on whether it is in its metabolically-inactive (i.e., endospore) or active (i.e., vegetative) state, as T cell differentiation was driven toward inflammatory, intracellular T H 1 responses and extracellular T H 2 responses via sporous and vegetative B. subtilis, respectively (138). Thus, de-sporulation in the intestine is a critical factor that could have major consequences on the host immune response.
GalliPro R consists of a single strain, B. subtilis DSM 17229 which improved performance, and reduced ammonia emission from the excreta in broilers (98). GalliPro R has been shown to reverse loss of splenic mass in Salmonella-infected birds, although no immune parameters were changed when non-infected birds were fed this probiotic (67). Furthermore, GalliPro R increased the liberation of crude protein from the diet, consequently decreasing broiler feeding costs and increasing body weight and feed conversion ratios (100). However, this study did not show whether GalliPro R was directly involved in this liberation or indirectly through a shift in the microbiota. Addition of GalliPro R to feed reduced Salmonella in cecum samples and greatly reduced Salmonella-positive drag swabs when compared to control broilers (99). Lastly, GalliPro R facilitates complete elimination of C. perfringens colonization in the ileum of challenged birds (101).
CloSTAT R contains a single strain of B. subtilis, PB6. When included to the diet of C. perfringens-challenged broilers at 1 × 10 9 CFU CloSTAT R /g feed, these broilers had statistically increased body weight and feed intake counts compared to challenged broilers without probiotics (139). However, CloSTAT R supplementation did not significantly change bacterial load of lactobacilli nor C. perfringens in the ileal digesta (139).When investigating the mortality rates from E. coli challenge comparing broilers fed CloSTAT R , control, and antibiotic growth promoters, CloSTAT R showed reduction comparable to the antibiotic growth promoter (both significantly compared to control) (103). Similarly to GalliPro R , CloSTAT R also reduced C. perfringens colonization of the ileum upon challenge (102).
Norum TM is a direct-fed microbial culture that consists of two B. amyloliquefaciens strains (AM0938 and JD17) Addition of Norum TM has shown an increase in productivity parameters like body weight, body weight gain and feed conversion (105,140). Norum TM greatly reduced the gut permeability and leakage of mucosal, immunological effectors like IgA into serum (105). In a necrotic enteritis model in which birds were challenged by Salmonella Typhimurium, Eimeria maxima, and Clostridium perfringens at days 1, 13, and 18-19 post-hatch, respectively, Norum TM significantly improved lesion scores (105). Lastly, in ovo administration of Norum TM to the feed greatly decreased the horizontal transmission of virulent E. coli and infection of broiler chickens during hatch, possibly through alterations of microbiota composition and community structure (108).

Bifidobacterium, Saccharomyces, and Multi-Species Probiotics
To the authors' knowledge, there are no commercial poultry probiotics solely-constituted of Bifidobacterium spp. However, Bifidobacterium spp. are widely used in combination with Lactobacillus probiotics (ex: PrimaLac R ) and other combination products (ex: MicroGuard R ). Bifidobacterium directly affects IgA secretion in the gut (141) as well as stimulates professional phagocytes and pancreatic elastase production via secretion of the serine protease inhibitor Serpin (142). This proinflammatory mechanism action suggests that Bifidobacterium Serpin-production is involved in the homeostasis of the gut microbiota. Additionally, Bifidobacterium spp. produces acetate and lactate, which are subsequently-used by microbial gut fermenters to produce butyrate and propionate (143). These two short-chain fatty acids (SCFAs) promote colonic regulatory T cell differentiation (144,145) as well as increase bactericidal functions of intestinal macrophages (146). Furthermore, the high GC content of the Bifidobacterium genome interacts with TLR9 that is present on the surface of mammalian immune cells (141,147), although it is not clear whether Bifidobacterium DNA has a similar effect on the avian analog TLR21 (148).
Although the scope of this review is live bacterial prophylactics, the eukaryotic Sacchormyces species S. cerevisiae and S. boulardii [although S. boulardii is arguably a sub-species of S. cerevisiae (149)] are widely-implemented in poultry probiotic mixtures (i.e., Gro-2-Max R and MicroGuard R , respectively) and thus will be briefly-mentioned. Despite these two species being highly-similar, S. boulardii has greater heat and acid tolerance vs. S. cerevisiae, making it more competitive in the gut microenvironment [reviewed in (150)]. Additionally, both Saccharomyces species increased SCFA production via shifts in the microbiome (151,152). Furthermore, S. cerevisiae and S. boulardii can directly-eliminate pathogens via secretory antimicrobials (153,154). However, only S. boulardii appears to possess membrane-associated inulin, which can agglutinate pathogens (155,156).
Lavipan R consists of several LAB (Lactobacillus casei LOCK 0915, Lactobacillus lactis IBB 500, Carnobacterium divergens S-1, and Lactobacillus plantarum LOCK 0862, all at 1 × 10 9 CFU/g product) and Saccharomyces cerevisiae LOCK 0141 (1 × 10 7 CFU/g) and was shown to competitively exclude pathogenic bacteria such as Campylobacter spp. and Salmonella Enteritidis (109). This probiotic also improved villi morphometric parameters (i.e., villus width and surface area) of the duodenum, jejunum, and ileum compared to control group (110). Lavipan R supplementation also caused reduced Clostridium spp. and Escherichia coli when compared to the control broilers, which was increased with the addition of prebiotics (i.e., raffinose family oligosaccharides) (96), which are non-viable food components like that improve host health via direct modification of the commensal microbiota (157). Thus, adding prebiotics to commercial probiotic products may improve health outcomes in poultry animals.
PrimaLac R is composed of Lactobacillus acidophilus, Lactobacillus casei, Enterococcus faecium, and Bifidobacterium bifidium, all at 1 × 10 6 CFU/g (158). The use of PrimaLac has been shown to limit the colonization of Salmonella and E. coli (111) as well as C. jejuni (115). However, this probiotic does not induce any changes in ceca lactobacilli (113,115). Supplementation of this probiotic to broilers in ovo produced an upregulation of iNOS, crucial for improving macrophagekilling of bacteria, in the ileum at day-of-hatch. However, later time points observed PrimaLac R -mediated downregulation of immune genes encoding toll-like receptors, cytokines, and iNOS in the ileum and ceca tonsil (116). The addition of PrimaLac R to the feed of turkey poults reduced Salmonella colonization upon challenge when compared to the control birds (111). When compared to an antibiotic growth promoter and control groups, addition of PrimaLac R increased reduction of C. perfringens, as well as improved broiler performance (114).
MicroGuard R contains 11 microorganisms (Bacillus licheniformis, B. megaterium, B. mesentricus, B. polymyxa, B. subtilis, Saccharomyces boulardii, Bifidobacterium bifidum, Lactobacillus acidophilus, L. bulgaricus, L. plantarum, and Streptococcus faecium) (159). The addition of MicroGuard R to the commercial broilers increased final bodyweight, weight gain, high density lipoprotein, triglyceride, and antibody titers against Newcastle disease and avian influenza levels (117). The addition of MicroGuard R also limited colonization of both Salmonella Enteritids and E. coli due to the above mentioned mechanisms, competitive exclusion, and possibly the production of bacteriocins (117).
Lastly, Gro-2-Max R is a multi-species probiotic product containing LAB (Lactobacillus acidophilus, Pediococcus pentosaceus, P. acidilactici), Bacillus subtilis, and Saccharomyces cerevisiae. When comparing route and length of treatment, Gro-2-Max R supplementation via food or water had general physiological impacts like reduced total triglycerides, lowdensity lipoprotein cholesterol, circulating lymphocytes, and viral vaccine-specific antibody titers. Additionally, ceca Enterobacteriaceae levels were inconsistently increased or decreased by Gro-2-Max R , regardless the route of inoculation (119). Our team has recently demonstrated changes in chicken intestinal Enterobacteriaceae levels via Gro-2-Max R , with layers only fed Gro-2-Max R exhibiting increased Enterobacteriaceae fecal shedding compared to the control birds (160). Furthermore, layers showed increased resistance to both APEC and Salmonella Kentucky when fed with both live Salmonella vaccine and Gro-2-Max R (118), suggesting this probiotic has adjuvant activities.

FUTURE DIRECTIONS FOR LIVE PROPHYLACTICS
Although much progress has been made in protecting poultry against bacterial disease, the movement of poultry animals to cage-free facilities has driven an increase in bacterial infections [reviewed by (3)], which pose risks to both animal and human health. Although the previously-described commercial vaccines and probiotics are used in practice, there are emerging technologies and strategies to improve food safety that warrant discussion. For the duration of this review, we will highlight issues in probiotic label-accuracy, novel yet non-commercial live vaccine strategies, and research gaps where the effects of probiotics and live vaccines are largely-understudied.

Probiotic Product Label Reliability
Although probiotics are widely-implemented in animal agriculture, label accuracy is a major concern that can drastically-influence product efficacy and health outcomes in poultry animals. More than 28% of the commercial cultures intended for human or animal use were misidentified at the genus or species level through rapid detection methods (161). Looking specifically at poultry probiotics, Redweik and colleagues use PCR to confirm the identification of all probiotic bacteria in Gro-2-Max R but detected Saccharomyces pastorianus (not S. cerevisiae as advertised) (160). Using four different methods to taxonomically-identify LAB present in FloraMax R -B11, Menconi and colleagues found that each method produced mixed results (93). Although 16S sequencing was the most accurate method used in this study, it is nearly impossible to speciate bacteria via 16S sequencing unless they are highly-characterized [reviewed in (162)], demonstrating its limited application. Using whole-genome shotgun sequencing is a far more accurate tool in addressing current labeling issues and false positive results for species not listed as components (163). Finally, another major concern is the accuracy of bacterial concentrations in these commercial products, as total viable cell counts often do not correspond with the concentrations given on the label (164). Altogether, it is imperative that researchers studying commercial probiotic activities in poultry verify label accuracy to improve repeatability.

Novel Live Vaccine Strategies
Although live vaccine technologies for Salmonella, APEC, Mycoplasma gallisepticum, and Pasteurella multocida are commercially-available for poultry animals, there is no commercial live vaccines for Campylobacter nor Clostridium available. Campylobacter, a major foodborne pathogen responsible for intestinal and extraintestinal disease in humans (165,166), typically colonizes the chicken gut as a commensal (167). Despite several studies evaluating the use of whole-cell Campylobacter vaccines (168)(169)(170)(171) and antigen-based vaccines (172)(173)(174)(175)(176)(177), there is no vaccine commercially-available for Campylobacter reduction in the intestine. A major issue with orally-delivered, live Campylobacter vaccines may arise in distinguishing between vaccine and pathogenic strains during meat processing. To avoid this issue, one solution could be to use another vaccine strain that is genetically-modified to express conserved Campylobacter antigens. Several studies have explored the use of Lactococcus lactis (178), Salmonella (179), and E. coli (180) to carry these antigens for anti-Campylobacter immune development. However, a major limitation to using antigen-based strategies against Campylobacter is that they are highly, antigenically-variable between strains (181), making the identification of a conserved target difficult.
Although necrotic enteritis is a major cause of mortality and reduced productivity in young birds (182,183), no vaccine is available against its causative agent Clostridium perfringens. Non-virulent C. perfringens can be used to promote intestinal immunity against pathogenic strains (184). Furthermore, Salmonella vaccines carrying recombinant C. perfringens antigens have been successful in potent protection against necrotic enteritis (185,186). Thus, there is much potential for a live, oral vaccine that can protect against C. perfringensinduced necrotic enteritis, which might be further-improved through support with probiotics like Cylactin , GalliPro R , and CloSTAT R which, on their own, offer protection (96,101,102).

Research Gaps
Most studies evaluate probiotic and live vaccine-efficacy by comparing mono-treated animals vs. non-treated controls. While this experimental design is a crucial first-step in identifying the usefulness of a live prophylactic, this format is not representative of natural commercial conditions and it ignores the impact other vaccines, feed, etc. may have on the animal's response to that live prophylactic of-interest. This is of extreme-importance, as commercial farms routinely use a wide repertoire of prophylactics (live, inactivated, and subunit alike) on their poultry animals without knowing how they might improve or nullify each other's effects. Probiotics are widely-reported to serve as biological, vaccine adjuvants [reviewed in (187)]. However, the role of probiotics in vaccine-responsiveness is largely-understudied in poultry. As mentioned briefly, efficacy and weight gain of a live, recombinant Campylobacter vaccine was drastically-improved in broilers which were also given Anaerosporobacter mobilis as a probiotic (180). This improvement in vaccine response is even found for live vaccines outside the scope of this review. The protection against the eukaryotic pathogen Eimeria was highest when a live coccidiosis vaccine was combined with probiotics (188). Use of Gro-2-Max in combination with a live Salmonella vaccine improved resistance to both intestinal Salmonella Kentucky colonization and extraintestinal infection by an O78 APEC (118). This latter study suggests that probiotics can even exert their benefits outside of the intestine, potentially through activation of immune phagocytes via TLR-dependent pathways (126). Another commercial probiotic (Cylactin R ) also may be a useful vaccine adjuvant, as combining this product with the live Salmonella vaccine Gallivac R SE increased Salmonella-specific IgA in layers (97). Thus, it is imperative that future studies look at the synergistic-effect other prophylactics may have on one another. Given the expensive nature of trying to fully-model the spread of prophylactics used in poultry agriculture, one could feasibly use commercially-available birds already given their respective prophylactics prior to experimental treatment.
Although parameters such as weight gain, food-conversion, egg laying efficiency, and bacterial resistance are commonlyused to study prophylactic-efficacy, there are many other mechanisms in which these live microbes could affect the host. Gut bacteria play a major role in the maturation of the enteric nervous system (189) and mediate animal behavior via the gutbrain-microbiota axis (190)(191)(192). These interactions are largelydriven by the ability of probiotic bacteria, Salmonella, and E. coli to directly synthesize and respond-to neurochemicals through a bidirectional communication network called microbial endocrinology (193)(194)(195). Animal models have demonstrated the ability for probiotics like Lactobacillus and Bifidobacterium (196,197) as well as C. perfringens (198) to modulate behavior, although only the latter has been shown in chickens. Recently, a 3761-derived Salmonella vaccine and Gro-2-Max were shown to modulate gut catecholamine (but not serotonin) metabolism in layer pullets, depending if the live prophylactics were given individually or in combination (160). Altogether, these findings suggest that the prophylactics used may have a direct impact on animal behavior. Thus, a novel target for live prophylactics could be to manipulate poultry animals into exhibiting positive behaviors (feeding, dust-bathing) while mitigating negative social behaviors like pecking. However, a major consideration is whether effects of these live prophylactics on the gut-brain-microbiota axis are maintained by chickens with different gut microbiotas. Given the variability of the chicken gut microbiome due to factors like geographical location, litter, breed, and feed [reviewed by (199)], it is very possible that other commensal bacteria might nullify, reduce, or amplify the effects live prophylactics might have on animal behavior and neurochemical metabolism.

CONCLUSIONS
Commercial live bacterial vaccines and probiotics offer several advantages in improving poultry health against bacterial disease and colonization (summarized in Figure 1) However, a paucity of peer-reviewed research studies, inconsistencies with product labels, limited cross-protection against certain pathogens, and a vague understanding of synergistic effects when using multiple prophylactics have encumbered our ability to optimize poultry health. Additionally, it is crucial that future studies must investigate whether these live prophylactics may facilitate animal behavior changes via the gut-brain axis (Figure 1), providing a convenient means of improving social behaviors among poultry flock.

AUTHOR CONTRIBUTIONS
GR and JJ wrote manuscript and developed figures and tables. MM revised manuscript. GR and MM provided funding and conceptualized review topic. All authors contributed to the article and approved the submitted version.

FUNDING
This review was supported by Iowa State University Start-up funding to MM and USDA-National Institute of Food and Agriculture (NIFA) project #021069-00001 to GR. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.