An Updated Review of Enterococcus cecorum Infections in Poultry

Enterococcus spp. are Gram-positive, non-spore–forming bacteria first identified in 1899 as the cause of fatal endocarditis (1). However, this genus includes more than 50 species predominantly of gastrointestinal tract origin in animals and humans (2). Enterococcus spp. possess unique mechanisms to enhance colonization, evade host immune defenses, and withstand harsh environmental conditions reviewed by Krawczyk et al. (3). The increase in Enterococcus-associated disease in both humans and livestock may be related to the acquisition of competitively advantageous virulence mechanisms and antibiotic resistance genes (4) or improved survival in the environment (5). The emergence of multidrug resistant bacteria, including Enterococcus spp., promoted a more judicious use of antibiotics by physicians and veterinarians. In humans, Enterococcus faecalis and Enterococcus faecium cause most enterococcal-related diseases, with E. faecalis being more frequently associated with endocarditis (6). However, there has been an increase in non-faecalis and non-faecium enterococci related bacteremia and endocarditis (7), including Enterococcus cecorum (EC), which has been irregularly associated with clinical disease in humans (8). In contrast, clinical disease and mortality have been frequently associated with EC infections in older chickens (9). In 1983, EC, previously classified as Streptococcus cecorum, was first isolated from the gastrointestinal tract of a healthy chicken (10). Although EC has been considered to be a commensal in the avian gastrointestinal tract, EC-related septicemia and lameness, first described in 2002 (11), has caused substantial economic losses for the poultry industry due to poor flock performance, increased mortality, and higher condemnation rates at processing plants (9,11,12,13,14). Previously, clinical disease was primarily observed in older chicken flocks, indicating that subclinical EC infections tend to go unnoticed until skeletal disease occurs. However, within the last few years, there has been an emergence of virulent EC strains associated with increased morbidity and mortality in young broiler chicken flocks (15). Recurrent outbreaks of lameness in affected houses in successive flocks have exacerbated the impact of this disease (16,17). It has been hypothesized that efficient gut colonization followed by translocation to extraintestinal sites, including the heart, spleen, bone, and cartilage, are critical factors of EC pathogenesis (18,19). The transmission dynamics of EC in commercial poultry operations have not been well defined nor has vertical transmission of EC from the broiler breeder hen to offspring been validated (12,13). The early onset of clinical disease associated with pathogenic EC-related septicemia in broiler chickens suggests that exposure may occur prior to placement at the farm, such as during late embryogenesis. Although not proven, the increased occurrence of virulent EC in young broiler chicken flocks could be a by-product of the discontinued use of antibiotics in hatcheries in 2019 (20). In-feed antibiotic usage in poultry has also significantly decreased within the last decade, and in some production systems, the use of antibiotics is not permitted (20). In addition, there are currently no approved vaccines to control diseases caused by enterococci in livestock (21), and vaccines specific to EC have been minimally investigated (22). Borst et al. (22) demonstrated that enterococcal spondylitis (ES) in broiler chickens was not prevented by administering an inactivated polyvalent EC to broiler breeder hens, suggesting that pathogenic EC strains possess virulence attributes that promote evasion of the host immune response, including an antibody-mediated response. Compared with nonclinical or commensal EC strains, pathogenic EC strains appear to more readily withstand exposure to harsh environmental conditions (23,24) and to survive exposure to host-derived antimicrobial compounds, such as lysozyme (25). To date, the routes and mechanisms of EC pathogenesis and transmission, especially related to the emerging pathogenic strains being detected in young flocks, have yet to be fully elucidated.

Historically, EC infections caused mortality in older broiler chicken flocks due to early septicemia, followed by lameness, resulting in dehydration and starvation of paralyzed birds (9). The traditional disease presentation includes two distinct phases: 1) the septic phase that occurs within the first 3 wk of life and 2) the skeletal phase that occurs in older flocks (26). Factors related to EC pathogenesis, such as intestinal colonization and bacteremia, have been shown to predispose chickens to ES (19,26). Anecdotal evidence from ES outbreak investigations has led to speculation that concurrent intestinal disease is needed for pathogenic EC to escape the gut and cause systemic infection (19). Although the exact translocation mechanism is unknown, it is thought that EC reaches the vertebra via hematogenous spread as a result of damage to the intestinal system, skin, or through the air sacs, as some vertebrae are pneumatic (27). Borst et al. (19) demonstrated that commensal EC strains colonize the gastrointestinal tract of chickens at 21 days of age, whereas pathogenic EC strains have been recovered from the gastrointestinal tract as early as 7 days of age. Pathogenic EC strains have also been isolated from the yolk sac or spleen as early as 14 days of age, suggesting bacterial translocation (18). The ability to colonize the gut early in life may provide pathogenic EC strains with a competitive advantage and potentiate dissemination throughout a flock. More recently, Arango et al. (15) demonstrated that EC can be isolated from the vertebrae at hatch, suggesting that exposure to virulent EC during embryogenesis could be linked to EC-associated disease in young broiler chickens. Pioneer colonization, which refers to these first microorganisms to colonize the gastrointestinal tract by noncommensal, opportunistically pathogenic bacteria such as virulent EC during the neonatal period, may increase the risk of disease in broiler chickens. More research is needed to define the role of EC pioneer colonization of the gastrointestinal tract and disease pathogenesis in broiler chickens.

Typically, flock history, macroscopic examination of lesions at necropsy, and microbiologic culture techniques are used to diagnosis diseases in poultry. Lesions following EC infection may vary, but a very typical and almost pathognomonic finding is ES and osteomyelitis at the level of the free thoracic vertebra (FTV) (9). In particular, during the septic phase, when lameness may be less obvious and the clinical manifestation may be minor (i.e. low mortality rate), the macroscopic lesions may be limited (9). Depending on the stage in the course of the infection, sampling from several of the predilection sites (joints, pericardium, and bone marrow) may increase recovery of pure EC (9). Although confirmation of clinical disease using the previously mentioned procedure provides strong evidence of EC infection, the absence of disease does not mean that the infection is not present because the septic phase generally presents as subclinical.

During the septic phase, subclinical or clinical disease may be observed. Affected flocks may be asymptomatic or show nonspecific symptoms such as depression, ruffled feathers, and retarded growth along with reduced performance and increased mortality due to systemic infection. Pathogenic strains of EC can be detected in the spleen and other organs as early as the first week of life, indicating early bacteremia and systemic spread (19). Flocks experiencing EC outbreaks have elevated mortality due to a combination of sepsis early in the growing period and dehydration and starvation of paralyzed birds late in the growing period (9). The typical course of the disease is characterized by a septic phase during the first 3 wk of the production cycle, followed by the skeletal phase, which lasts from week 3 until the end of the production cycle (26).

Postmortem findings may reveal marked pericarditis, which involves inflammation of the pericardium and the formation of fibrinous exudate around the heart, affecting its function and increasing mortality rates (18). Hepatitis may also be observed with affected livers showing signs of inflammation, hepatomegaly, and multifocal necrotic lesions, disrupting normal liver function and contributing to the systemic spread of the infection (12). Splenomegaly may be noted perhaps with a mottled appearance due to multiple areas of necrosis, indicating a severe systemic immune response, as the spleen attempts to filter out circulating bacteria, with EC being frequently isolated from the liver, heart, or spleen (12,18). Focal areas of necrosis on the myocardium may be observed, and histologically, heterophilic, histiocytic, and lymphocytic epicarditis have been described (18). Differential diagnosis is often required, considering the overlap between septicemia associated with EC and other opportunistic pathogens, such as Escherichia coli.

Bacteremia associated with the septic phase facilitates the translocation of pathogenic EC from the gastrointestinal tract to skeletal structures (19). During the skeletal phase of EC infection in broiler chickens, the bacteria primarily affect the FTV and the femoral heads (28,29). The first signs of lameness mark the onset of the skeletal phase of disease, and affected birds suffer from progressive ataxia due to development of an inflammatory mass at the FTV (28). Affected birds are lame or completely paralyzed and often found in a sitting position on their hocks with both legs extended to the front (30). Mortality during the skeletal phase typically peaks toward 6 wk of age (31). EC is frequently isolated from inflammatory lesions at the FTV or the femoral or tibial heads (32). Pathogenic EC translocates from the intestinal tract into the bloodstream, facilitating its spread to skeletal structures (19). In the FTV, bacterial colonization leads to osteomyelitic lesions characterized by inflammation and necrosis of the bone and bone marrow, causing deformities and locomotor issues (19). In the femoral heads, hematogenous opportunistic bacteria colonize microfissures, leading to osteomyelitis and cartilage necrosis. These infections cause severe lameness and can make the femoral heads fragile and prone to fractures (33,34). Osteomyelitis manifests as pus-filled cavities within the bone and surrounding tissues in predisposed joints of the spine and legs, leading to lameness and paralysis (28,29).

Arthritis may also occur, causing joint inflammation, swelling, pain, and reduced mobility, posing significant animal welfare concerns (9). In severe cases, EC can lead to endocarditis, an infection of the heart valves, indicating an advanced stage of systemic infection (12). These extensive extraintestinal lesions underscore the invasive nature of EC and its ability to cause widespread systemic disease, highlighting the importance of the intestinal microbiota in bone health and the potential dissemination of systemic infections from the intestine to other organs (33).

The routes of EC transmission in commercial poultry operations have not been well defined. Evidence suggests the increased incidence of enterococcal-associated disease in poultry may be due to horizontal spread of dominant clones of EC that exhibit increased pathogenicity (12,35). Vertical transmission from hen to progeny has been considered, but studies have not conclusively demonstrated genetic similarity between parent breeder strains and pathogenic outbreak strains in progeny flocks (12,14). Horizontal transmission of EC, especially during late embryogenesis, could be a critical period for transmission due to the rapid onset of morbidity and mortality in young broiler flocks and the resilience of EC in the environment (9,23). Once established in the environment, horizontal transmission of EC appears to occur rapidly in affected flocks, and the primary route of infection is believed to be fecal-oral, as intestinal infection typically occurs prior to development of other lesions of infected chickens (19). More recently, Arango et al. (15) demonstrated that pioneer colonization by virulent EC when administered by in ovo injection during late embryogenesis, promoted persistent colonization of extraintestinal organs, including the FTV, and hindered 21-day performance in broiler chickens, indicating that early exposure to virulent EC prehatch can lead to early colonization of the gastrointestinal tract, providing pathogenic EC strains a competitive advantage over commensal bacteria. This early colonization may result in systemic spread of EC and persistence of the infection, leading to significant health issues and decreased performance in broiler flocks (36). Olsen et al. (36) reported that the same sequence type of E. faecalis recovered from broiler chicks at hatching was reisolated at a higher incidence 24 hr posthatch, indicating that circulating Enterococcus spp. in the hatcher could influence the pioneer colonization of the gastrointestinal tract in neonatal broiler chicks. However, to date, there have been no studies evaluating horizontal transmission of EC during the hatching phase. This early colonization may provide pathogenic EC strains with a competitive advantage and enhance dissemination throughout the flock. The increased incidence of EC-associated mortality in young broiler chicken flocks requires further investigation because EC contamination in the hatchery could lead to sufficient colonization in naïve chicks before transfer to or placement at the farm.

Enterococcus spp. are inherently resistant to environmental stressors, including desiccation and temperature changes (37), suggesting that EC could reside in the hatchery or barn environment for extended periods. However, there is limited research on EC resiliency in the environment. Studies have shown that EC can survive at low temperatures and even withstand exposure to 60 C for up to 1 hr (38) and that the type of matrix, such as litter, dust, or polyvinyl chloride (PVC) may differentially affect EC survivability in the environment (23). For instance, EC survival was extended for 1992 hr (83 days) in litter compared with dust or PVC (23). Worth noting, virulent EC strains were more resilient than commensal EC strains when subjected to suboptimal environmental conditions (23). Similar findings were described by Watson et al. (24) who demonstrated that a clinical EC isolate recovered from joint fluid was more resilient to desiccation and an increased environmental temperature (23 vs. 32 C) than EC recovered from the ceca of a healthy chicken. This indicates that survivability and transmissibility of EC could be correlated to virulence mechanisms, but additional research is needed to elucidate this. Evidence of persistent EC contamination in the environment, even after cleaning and disinfection practices have been implemented, indicates the significant risk of cross contamination between flocks (39). In addition, detection of EC throughout the production cycle can be difficult when other Enterococcus spp. are present, which may require using unique approaches, including chromogenic media supplemented with antibiotics, to improve EC detection (39). Further research to define points of exposure to EC and transmission within a flock is needed to develop methods to control EC contamination and transmission in the field. At present, implementing strict biosecurity measures, including thorough cleaning and disinfection protocols, and managing water and feed quality are the most crucial strategies to reduce transmission and cross contamination of EC on poultry farms.

Methods used to detect and differentiate between Enterococcus spp. with a particular focus on EC may include primary culture and phenotypic evaluation, followed by genotyping using multilocus sequencing, pulsed-field gel electrophoresis (PFGE), 16S ribosomal RNA sequencing, or species-specific PCR. Refer to a review published by Jung et al. (9) for a more comprehensive summary of detection methods for EC.

In brief, EC presents as a mucoid greyish colony with marginal alpha-hemolysis and Gram stains as Gram-positive cocci in pairs and short chains. Colony size is typically 2–3 mm but smaller colonies, >1 mm, have been described (15,28). Tryptic soy agar supplemented with 5% defibrinated sheep blood (blood agar) can be used for cultivation of EC when inoculated plates are incubated 18–24 hr at 37 C under microaerophilic conditions (38). Chromogenic agars, such as CHROMagar Orientation, expedite the identification of some Gram-negative bacteria and some Gram-positive bacteria on the basis of different contrasted colony colors produced by reactions of genus- or species-specific enzymes with a proprietary chromogenic substrate (40). Enterococci are chromogenically distinct on CHROMagar Orientation agar. The two most commonly encountered species, E. faecalis and E. faecium, have similar color reactions and are indistinguishable at the species level (40). EC also cannot be distinguished between other enterococci on CHROMagar Orientation. Thus, enterococci-like colonies must be further identified using methods such as basic phenotypic tests, matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS), or PCR (28,38).

For most clinical microbiologic laboratories, the primary method of identifying Enterococcus strains relies on phenotypic characterization using commercial test kits (41). However, such identification may cause problems with poultry isolates, because they may differ slightly in phenotypic profiles compared with the human isolates used to develop the identification schemes and databases. Studies have shown that unequivocal species identification of Enterococcus by phenotypic means is a challenging procedure that can take several days to accomplish because of the phenotypic and biochemical similarities between many species of Enterococcus (41). Dolka et al. (38) used API Rapid ID 32 STREP and the Biolog System to determine and differentiate between biochemical and metabolite profiles of EC recovered from poultry. PCR methods, including multiplex PCR targeting sodA (42) and real-time PCR (43) have been used for detecting EC.

An alternative way to differentiate species within the same genus is by phyloproteomic analysis of the isolates’ spectral profiles by MALDI-TOF MS (44). MALDI-TOF MS can be used for preliminary analysis and comparison of isolates, including differentiation of closely related Enterococcus spp. based on protein profiles (44). PFGE, a method for digesting genomic DNA resulting in a unique banding pattern using an agarose gel (45), has also been used to differentiate between Enterococcus spp. and between isolates of the same species, specifically EC (14,46).

Commensal bacteria are those that can act on the host’s immune system to induce protective responses that prevent colonization and invasion by pathogens, whereas pathogenic bacteria can harm and cause infectious disease. Enterococci are generally opportunistic pathogens (3). However, EC strains have been categorized as pathogenic when recovered from extraintestinal organs (29).

Embryo lethality assays (ELAs) have also been used to characterize the virulence of EC strains (47,48,49,50). Extraintestinal EC isolates seemed to cause higher embryo lethality rates, appearing more virulent than stains derived from the gastrointestinal tract (49). However, EC strains that were deemed pathogenic using ELAs did not cause disease when administered orally at day of hatch (29), contradictory to findings described by Borst et al. (19). There is limited information available regarding the virulence of EC isolates recovered from animals other than broiler chickens (28). In this article, the authors demonstrated that pathogenic and commensal EC isolates from different animal species (chicken, Pekin duck, turkey, pigeon, cattle, swine, budgerigar, goose, human, Muscovy duck, swan) vary in virulence when evaluated using a chick embryo lethality assay and also differed in the ability to metabolize mannitol. In this study, embryo mortality caused by pathogenic EC isolates was 39.7%, which was significantly higher than the 18.9% mortality associated with commensal EC isolates. In addition, the authors showed that EC isolates recovered from spinal lesions possessed a decreased ability to use d-mannitol compared with cecally derived isolates. Furthermore, genes related to mannitol metabolism were detected in nonpathogenic EC isolates but were absent or nonfunctional in more pathogenic strains, implying that mannitol utilization could discriminate between virulent and avirulent EC strains.

As mentioned previously, PFGE has been used to identify EC (14,46). When comparing presumably commensal and pathogenic EC strains, PFGE showed that EC strains recovered from extraintestinal organs (pathogenic) were genetically diverse from intestinal strains (commensal), but multiple unique banding patterns within the pathogenic strains evaluated complicate PFGE results (14). Genomic analysis further indicated that pathogenic EC strains were divergent from commensal strains (51). More recently, a comparative genomic study evaluating clinical EC strains isolated in France showed that clinical strains are phylogenetically distant from nonclinical isolates (52). The investigators also noted that the clinical EC isolates from France were similar to those that have been described in North America.

Small colony variants (SCVs) further complicate identification and differentiation of EC strains. SCVs constitute a slow-growing subpopulation of bacteria with distinctive phenotypic and pathogenic characteristics (53). Since their first description in 1910, SCVs have been described in a wide range of gram-positive and gram-negative bacterial species, including Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Salmonella enterica, Pseudomonas aeruginosa, and Burkholderia cepacia (54). SCVs seem to be able to persist intracellularly and to be less susceptible to antibiotics (54), causing latent and recurrent infections. The low growth rate and other unusual phenotypic characteristics make them a challenge for clinical microbiologists to identify (55). Evidence suggests that SCVs of E. faecalis may be associated with outbreaks of amyloid arthropathy in chickens (55) and endocarditis in humans (53). EC SCVs were first documented and evaluated for virulence by Jung et al. (28). These EC SCVs appeared to be apathogenic compared with EC that had a normal colony morphology but were isolates obtained between 1995 and 2001. A pathogenic EC SCV that causes septicemia and macroscopic heart lesions in young broiler chickens has been recently described (Fig. 1) (15). Additional studies are needed to provide a more comprehensive understanding of the frequency and pathogenicity of EC SCVs in poultry.

Virulence factors are components that may be surface associated or secreted and promote the establishment of a pathogen in a host. The variability in virulence among EC isolates is significant, with certain strains causing more severe disease manifestations than others (15,19,29,38,48,49,56,57). Pathogenic EC strains possess virulence factors that allow them to adhere to host cells, invade tissues, and evade host immune responses. Genotypic tests, including PCR (19,38,56,57) or genomic analysis (51,52,58,59) can be used to determine if the operon or genes of a virulence factor is present in EC. The presence of the gene does not necessarily indicate functionality or expression. If possible, phenotypic assays can be used to assess if a gene is functional (38,52,57). Virulence can be further assessed using in vivo models, such as embryo lethality assays (48,49,57,60,61). Enterococci possess certain cell surface components, including adhesions, pili, and a polysaccharide capsule associated with enhanced virulence (62). The polysaccharide capsule is an effective cell wall–associated structure that enables resistance to host immune defenses and degradation when exposed to stressors (62). Genomic analyses showed that the presence of capsular polysaccharide loci appears to be increased in clinical vs. commensal EC strains (51,52,59), indicating that the capsule plays an important role in EC virulence. Additional Enterococcus-associated virulence genes previously evaluated in both commensal and clinical EC strains have included asa1 (aggregation substance), gelE (gelatinase), hyl (hyaluronidase), esp (enterococcal surface protein), cylA (cytolisin), efaA (endocarditis antigen), ace (collagen-binding protein), epa (enterococcal polysaccharide antigen), efaAfm (cell wall adhesin of E. faecium), efaAfs (cell wall adhesin of E. faecalis), and ccf (sex pheromone) (28,38,51,56). In some studies, enterococci-related virulence genes have been detected in most clinical EC isolates evaluated (9,51). Overall, the detection of enterococcal virulence factors in clinical EC strains has been variable across studies, and in some cases, selected genes were not detected in any of the clinical EC isolates (38). The disparity between studies could be related to the number of EC isolates evaluated or may indicate pathogenesis is linked to factors unique to EC, such as the capsular loci, epa, or host-binding proteins (51) that would not be identified using primers targeting virulence factors designed for E. faecalis or E. faecium. As a result, whole-genome sequencing (51,52,59) has been used to pinpoint genes uniformly present across clinical EC isolates that could be related to virulence across clinical EC isolates.

Gene function or expression can be evaluated with phenotypic assays. There have been multiple studies in which genotype and phenotype do not align (38,57,63). For example, the gelatinase gene, gelE, has been detected in clinical EC and E. faecalis strains using PCR but was not correlated to gelatinase activity in vitro (38,63). This suggests that the presence of a virulence gene that lacks functionality may indicate that the gene is truly nonfunctional or that the conditions of the phenotypic assay do not permit expression of the gene. Mannitol use by commensal EC strains, but not virulent EC strains, has been frequently demonstrated (15,28,38,51), although gene presence and functionality in commensal strains may be variable (28). In addition, the ability to form biofilms (23,64), to adhere to collagen (65), or to survive in chicken serum have been shown to enhance virulence of enterococci (66), but these phenotypes were not consistently observed in the >100 clinical EC isolates evaluated by Laurentie et al. (52). Because the majority of the clinical EC isolates described by Laurentie et al. (52) were recovered from a joint, a more comprehensive comparative genomic analysis that includes more isolates recovered from other extraintestinal sites, including the heart, would be of interest. Accessory genes identified by predictive analysis (52) related to metabolic function specific to clinical EC isolates, mostly from affected joints, could be further investigated by incorporating isolates recovered from other tissues and from recent cases in young broiler chicken flocks. More recently, virulent EC strains were shown to exhibit greater resistance to lysozyme, a key antimicrobial component in egg whites and mucous secretions, which may increase survival during embryonic development (25). More research is needed to characterize virulence factors specific to EC pathogenesis.

Although antibiotics cannot be used in antibiotic-free production systems, antimicrobials can be used to treat bacterial infections in conventional production systems. Antibiotics have been used to treat EC infections detected early in chickens (9) but may be ineffective in clinically affected flocks. The tropism of EC for cartilage and pericardial tissues, when antibiotic concentrations may be insufficient, may contribute to the persistence of infections (12). In addition, intrinsic and acquired mechanisms of antibiotic resistance in Enterococcus spp. may complicate treatment regimens (67,68). Antibiotic resistance can be assessed using genotypic and phenotypic approaches, with the disk diffusion technique being preferred over broth dilution assays when evaluating resistance in EC isolates (61). Pathogenic EC strains have shown greater resistance to antibiotics such as tetracycline, oxytetracycline, and erythromycin (9,58), aligning with the continued use of tetracycline in conventional poultry flocks today. Recently, clinical EC strains proved to be susceptible to antibiotics relevant to humans and to antibiotics used in conventional poultry production (61). In 2012, vancomycin resistance was detected in EC isolated from chicken meat in Japan (69) but was not observed in a recent study evaluating 208 EC strains (61). However, in a more recent study, Huang et al. (70) reported vancomycin resistance in 50% of the nonclinical EC isolates but only in 4% of the clinical EC isolates. Multidrug resistance has also been reported, being more common in pathogenic EC strains compared with commensal EC strains (15,38,71), but resistance patterns do not appear to be consistent across isolates (71). Interestingly, resistance to antibiotics not used presently in poultry flocks has been observed and thought to be related to coselection that may occur when an approved antibiotic is used (61). Surveillance of antibiotic resistance patterns in EC strains of poultry origin is critical to identify epidemiologic trends associated with resistance, especially for those antimicrobials relevant to humans.

EC has emerged as a significant pathogen in chickens, with septicemia being more recently reported in young broiler chicken flocks. EC transmission and persistence in a flock is currently unknown, and virulence attributes detected in pathogenic EC have been somewhat variable across studies. Thus, understanding transmission dynamics using horizontal transmission models and detailed studies of virulence factors is crucial for developing effective control measures. The increasing prevalence of pathogenic EC strains in poultry poses a significant challenge for producers because the infection not only affects the health and welfare of broiler chickens but also has a considerable economic impact due to reduced performance and livability. Management strategies should include preventive measures such as improved biosecurity and the development of alternative treatments to mitigate the spread of EC. Vaccinating broiler breeder hens with an inactivated EC bacterin did not prevent clinical EC in offspring (22). Pathogenic EC strains have evolved mechanisms to evade the host immune response, including antibodies, which complicates vaccine development. Effective control strategies for EC infections in poultry must consider both direct and indirect routes of transmission, particularly during late embryogenesis.

Abbreviations:

EC =

Enterococcus cecorum;

ELA =

embryo lethality assay;

ES =

enterococcal spondylitis;

FTV =

free thoracic vertebra;

MALDI-TOF MS =

matrix-assisted laser desorption–ionization time-of-flight mass spectrometry;

PFGE =

pulsed-field gel electrophoresis;

PVC =

polyvinyl chloride;

SCV =

small colony variant