Enterococcus faecalis organisms are Gram-positive cocci that are ubiquitous in the environment, occurring in water and soil, and are commensal inhabitants of the intestinal tracts of both vertebrate and invertebrate animals. They are considered opportunists and possess many virulence-encoding traits, including the formation of biofilms and toxins. Enterococcus faecalis produces cytolysin, which is a unique toxin having activity against both eukaryotic and prokaryotic cells. Cytolysin causes hemolysis of red blood cells and has also been termed hemolysin. Enterococcus faecalis organisms are intrinsically resistant to some antibiotics and can transmit antimicrobial resistance to other microorganisms. In poultry, there is ample evidence to indicate that E. faecalis can be egg transmitted, causing decreased hatchability of eggs. Enterococcus faecalis has been found to rapidly spread among hatchlings that are exposed in the hatcher. In older birds, some E. faecalis isolates cause amyloid arthropathy. Various methods have been developed to assess E. faecalis isolates for virulence-encoding traits, including Multilocus sequence typing and embryo lethality assays. However, much variability occurs in interpreting the results of these methods and the correlation between genotypes, phenotypes, and virulence has not been well established. The virulence traits and pathogenesis of E. faecalis in poultry need to be elucidated further.

Revisión de las infecciones causadas por Enterococcus faecalis en la avicultura comercial.

Los organismos Enterococcus faecalis son cocos grampositivos que se encuentran en todas partes en el medio ambiente, en el agua y en el suelo, y son habitantes comensales de los tractos intestinales de animales vertebrados e invertebrados. Se los consideran oportunistas y poseen muchos rasgos que codifican para virulencia, incluida la formación de biopelículas y toxinas. Enterococcus faecalis produce una citolisina, que es una toxina única que tiene actividad contra células eucariotas y procariotas. La citolisina causa hemólisis de los glóbulos rojos y también se la ha denominado hemolisina. Los organismos El Enterococcus faecalis es intrínsecamente resistente a algunos antibióticos y pueden transmitir resistencia antimicrobiana a otros microorganismos. En la avicultura comercial, existe amplia evidencia que indica que E. faecalis puede transmitirse a través del huevo, lo que provoca una disminución de la incubabilidad del huevo. Se ha descubierto que Enterococcus faecalis se propaga rápidamente entre los pollitos recién eclosionados que están expuestos en la nacedora. En las aves de mayor edad, algunas cepas de E. faecalis causan artropatía amiloide. Se han desarrollado varios métodos para evaluar las cepas de E. faecalis en busca de características que codifican la virulencia, como la tipificación de secuencias de locus múltiples y los ensayos de letalidad embrionaria. Sin embargo, existe una gran variabilidad en la interpretación de los resultados de estos métodos y no se ha establecido bien la correlación entre genotipos, fenotipos y virulencia. Es necesario dilucidar más los rasgos de virulencia y la patogénesis de E. faecalis en la avicultura.

The taxonomic classification of Enterococcus faecalis is as follows: Phylum: Firmicutes, Class: Bacilli, Order: Lactobacillales, Family: Enterococcaceae. Genus: Enterococcus, Species: faecalis. Enterococcus faecalis cells are gram-positive, catalase negative, ovoid cocci occurring as singlets, pairs, or short chains (Fig. 1) (1,2,3). Enterococcus faecalis was originally classified as serological (i.e., Lancefield) group D Streptococcus faecalis but later transferred to the genus Enterococcus based on molecular analysis (2,4). Therefore, some early works may refer to E. faecalis as a streptococcus, especially if the organism was identified based on Gram staining and microscopic appearance. Using fossil records and molecular techniques along with environmental distribution analysis, the origin of enterococci species has been estimated to be 425–500 million years ago, occurring in parallel with the terrestrialization of animals (5). “Enterococcus” was coined by Thiercelin in the 19th century when he observed a gram-positive coccus originating from the intestines and capable of causing infection (6). Enterococcus faecalis is considered a ubiquitous microorganism that can be found in fresh water, seawater, soil, plants, animals, and food and is part of the microbiota of vertebrates and invertebrates (2). In humans, enterococci are some of the earliest bacteria to colonize the intestinal tract and are considered commensal organisms of the human intestinal microbiota (6) comprising less than 1% of the total intestinal microbiota (2,5).

Fig. 1.

Gram stain of E. faecalis, 1200×.

Fig. 1.

Gram stain of E. faecalis, 1200×.

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Early on, those involved with human medicine considered Enterococcus species ubiquitous organisms that were harmless and caused no disease or concern (3). However, Enterococcus species are now recognized as a leading cause of nosocomial infections leading to bacteremia, urinary tract infections, hepatobiliary sepsis, endocarditis, surgical wound infections, neonatal sepsis, and endodontic disease, which are often associated with catheters and implanted medical devices (2,3,6,7). Enterococcus faecalis is one of the most abundantly identified Enterococcus species involved in the aforementioned infections (2,8). Additionally, Enterococcus species have been identified as a leading cause of multidrug resistance in nosocomial infections, with E. faecalis being a major contributor (3,6,9).

Several virulence factors have been attributed to the pathogenesis of diseases caused by E. faecalis. Several publications can be reviewed for a more complete assessment of these virulence factors (2,3,6,10,11,12,13). Here we review those virulence traits believed to play a role in poultry diseases. These reported virulence traits (2,14,15,16,17) and their associated genes are summarized in Table 1.

Table 1.

Enterococcus faecalis virulence traits and associated genes.

Enterococcus faecalis virulence traits and associated genes.
Enterococcus faecalis virulence traits and associated genes.

Enterococcus faecalis infections are postulated to be endogenous whereby the organisms establish themselves in the gastrointestinal tract and then translocate via intestinal epithelial cells, are taken up through the lymphatic system, and then spread to other areas of the body through the blood stream (3,18,19,20). Dysbiosis, a condition whereby the intestinal microbiota is altered, can be caused by numerous conditions, including inflammation, infectious diseases, immune status, antibiotics, dietary changes, and stress (21). When dysbiosis occurs E. faecalis may readily propagate (i.e., overgrow) and translocate across the intestinal epithelial barrier (22). Additionally, it has been demonstrated that E. faecalis can enter intestinal epithelial cells and propagate within them (23,24,25). Enterococcus faecalis can survive within macrophages, and macrophages may provide a mode of transport for dissemination (23).

As mentioned above, Enterococcus species were originally classified as a Lancefield group D Streptococcus species. Following the reclassification of Enterococcus classification schemes were proposed based on the serological testing against the capsular polysaccharide. One of the earliest schemes proposed was by Maekawa et al. in which 42 E. faecalis strains were evaluated, and this resulted in proposing a classification scheme consisting of 21 serovars (26). Subsequent work by Hufnagel et al. reclassified E. faecalis into four capsular polysaccharide groups (CPS-A, CPS-B, CPS-C, and CPS-D) based on enzyme-linked immunosorbent assays and opsonophagocytic assays (27). To the authors’ knowledge, there are no reports of these classification schemes being used with E. faecalis isolates of poultry origin. Other microbial surface components recognize adhesive matrix molecules and are elements that assist E. faecalis in initiating and establishing infections (2,28). One protein, termed aggregation substance, aids the bacteria to amplify in number, colonize and overcome host defenses by limiting opsonization (6,12).

Another important virulence factor is cytolysin, often referred to as hemolysin (and streptolysin in the early literature) (2,6,16). Cytolysin is active against both prokaryotic and eukaryotic cells (6,10,16). It is a pore-forming exotoxin that lyses cells and responds via quorum signals (13,16). As a bacteriocin, cytolysin aids the bacteria in establishing colonization by impeding the growth of competing gram-positive bacteria (2). Cytolysin also acts against eukaryotic cells where it is toxigenic and plays an important role in tissue damage and increasing lethality (2,6,10). Using animal models, it has been shown to be associated with virulence (29). The cytolysin is a complex molecule encoded by eight genes and is expressed as two molecules whereby the smaller molecule is involved in quorum signaling (2,3,6,10,16,30). Cytolysin can lyse erythrocytes although susceptibility of erythrocytes to cytolysin varies substantially depending on the animal species. It has been reported that hemolysis is observed when E. faecalis is cultured on agar prepared with either horse, human, dog, rabbit, or mouse erythrocytes but not with sheep, cow, or goose erythrocytes (16,31,32). It has been the authors’ experience that chicken erythrocytes are not sensitive to hemolysis by E. faecalis, and sheep erythrocytes are less sensitive than horse erythrocytes (33). In a study involving 43 E. faecalis isolates from poultry in Portugal, cytolysin genes were detected in 40 of the isolates, but only one isolate demonstrated beta-hemolysis (34).

Enterococcus species can form biofilms by the cross-linking of pili. Enterococcus faecalis has also been reported to promote biofilm formation in mixed bacterial infections (35). Biofilms are an important virulence factor in urinary tract diseases, endocarditis, endodontic infections, and those infections originating from catheters and medical devices (2,3,6). Enterococcus faecalis can produce gelatinase, an enzyme that degrades collagen, gelatin, and lamin and plays an important role in biofilm formation, while also providing the bacterium a nutritional source and inhibits the complement-mediated host response (2,3,6). Antimicrobial resistance and reduced susceptibility to antibiotics is another important virulence factor that has caused major concerns with Enterococcus species infections (2,3,6). Enterococcus species are intrinsically resistant to cephalosporins, aminoglycosides, lincosamides, and streptogramins (6). Acquired antimicrobial resistance between Enterococcus species and other bacterial pathogens occurs via mobile elements such as pheromone-mediated conjugative plasmids or transposons (2,3,6,36). Briefly, pheromone-mediated conjugation is a complex process in which a plasmid-free bacterium emits small peptides (i.e., pheromones) that “attract” or signal those bacteria containing plasmids to conjugate and transfer the plasmid (37). Enterococcus faecalis has been reported to be a predominant bacterial species isolated from poultry meat (38). Because some virulence genes from E. faecalis of poultry origin are shared with isolates of human origin, there is a concern regarding zoonotic potential (39,40).

Overview

As stated above, Enterococcus species are ubiquitous and occur in soil and water and are a part of the intestinal microbiota of many animal species. Some Enterococcus species have been used as probiotics in poultry (41,42,43), and a strain of E. faecalis has been reported to have a positive effect on growth and immune function when fed as a probiotic (44). However, because of the concerns that Enterococci species can acquire and transmit antimicrobial resistance, their use in probiotic formulations should be carefully evaluated (45). Because of the ubiquitous nature of E. faecalis and because it is an opportunist, it is difficult to ascertain its role as a primary pathogen in many poultry disease conditions. For example, E. faecalis was isolated in cases of colibacillosis attributed to pathogenic Escherichia coli (E. coli), and it was found that E. faecalis augmented the growth of E. coli and increased the virulence when coinfected with E. coli into an embryo lethality assay (46). In this study, 12-day-old chicken embryos were inoculated with either monocultures of E. faecalis isolates or E. coli isolates or mixed cultures of E. faecalis and E. coli by the allantoic route, and the embryos were observed for mortality and lesions. In another independent study involving the coinfection of E. coli and E. faecalis isolates from broiler chickens, the monocultures and mixed cultures were grown in broth cultures, and 12-day-old chicken embryos were dipped into the bacteria containing solutions (47). It was reported that E. faecalis exposure resulting from the monoculture had no apparent negative effects on the embryos. However, with the mixed culture inoculum an increased (i.e., potentiated) virulence of E. coli was attributed to E. faecalis. An early report related that a case of septicemia in broiler breeders that led to increased mortality and valvular endocarditis where a Streptococcus species was isolated also identified E. faecalis on 16S ribosomal ribonucleic acid (rRNA) sequencing (48) as a contributor to the disease (see below). Another early report associated a streptococcus microorganism with a malabsorption enteric disease of chicks. This report concluded that a filterable agent was involved; however, antibiotics improved, but did not eliminate, the condition (49). Here we review those reports that relate E. faecalis disease conditions in poultry, the methods that explore E. faecalis virulence traits, and potential mechanisms by which E. faecalis causes disease in birds.

Hatchery and early life

One of the earliest reports of E. faecalis inhabiting the intestinal flora of young poultry was made by Devriese et al. (50) whereby the authors reported finding E. faecalis, E. faecium, and Streptococcus species in 1-day-old chicks but rarely found E. faecalis in 3- to 5-wk-old broilers. A report by Dolka et al. (51) conveying the occurrence of Enterococcus species isolated from poultry in Poland found seven species of Enterococcus occurring in broiler chickens, commercial layers, broiler breeders, and other poultry, including turkeys, ducks, and geese. The most frequently isolated Enterococcus species were E. faecalis (57%) followed by Enterococcus cecorum (7%), Enterococcus faecium (5.2%), Enterococcus hirae (3.6%), Enterococcus gallinarum (2.5%), Enterococcus casseliflavus (0.7%), and Enterococcus durans (0.2%). Within each poultry species, E. faecalis was the most frequently isolated Enterococcus species. The mean age of birds from which E. faecalis was isolated was as follows: broilers, 2.1 days; broiler breeders, 84.4 days; commercial layers, 54.2 days; turkeys, 28.2 days; ducks, 1.4 days; and geese, 124.3 days. An investigation of mortality within the first week of life in layer chickens revealed bacterial infection accounted for 50% of the mortality whereby E. coli and E. faecalis were identified as the most significant bacterial pathogens (52). Another early report by Alaboudi et al. (53) reported mixed infections of bacteria isolated from dead-in-the-shell chicken embryos. Those bacteria isolated may have potentially included E. faecalis. However, it is difficult to definitively determine if E. faecalis was isolated because they reported isolating “streptococcus.” The first definitive account of E. faecalis being isolated from dead embryos and causing infertility in pheasant eggs occurred at the 1993 North Central Avian Disease Conference by Reynolds and Akinc (54). At the time the bacterium was reported as a Streptococcus species based on microscopic evaluation. It was later identified as E. faecalis (Reynolds, pers. comm.). A similar finding was reported decades later by Reynolds and Loy (55) in a case report in which E. faecalis was isolated from late-stage dead-in-the-shell pheasant embryos that had pipped through the shell but did not hatch (Fig. 2). In this case, the hatchability suddenly dropped from about 75% to approximately 15%. No intervention strategies were employed, and within a few weeks the hatchability returned to its normal expected level. No predisposing factors could be linked to the reduced hatchability; however, there was an unseasonably cold and wet period that corresponded to when those eggs that experienced a decrease in hatchability were laid (note that pheasant breeders are housed in outside pens).

Fig. 2.

Unhatched ring-neck pheasant eggs. Note how eggs are pipped but embryos were unable to hatch. Enterococcus faecalis was isolated from the egg surfaces and from the internal organs of the embryos.

Fig. 2.

Unhatched ring-neck pheasant eggs. Note how eggs are pipped but embryos were unable to hatch. Enterococcus faecalis was isolated from the egg surfaces and from the internal organs of the embryos.

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The aforementioned reports provide clinical case data and circumstantial evidence for vertical transmission. An interesting study conducted by Tankson et al. (56) whereby broiler embryos and chicks were cultured for the presence of bacteria found that a low percentage of embryos had bacteria in their heart and lungs and the percentage increased following hatch. Although several bacterial species were isolated, the most frequently isolated species was E. faecalis. Proof of vertical egg transmission under experimental conditions was reported by Landman et al. (57), who intravenously injected adult laying hens with an arthropathic and amyloidogenic strain of E. faecalis and demonstrated chronic bacteremia and arthritis in infected birds. Enterococcus faecalis was isolated from the joints, ovaries, and oviducts of infected birds and from infertile eggs and dead embryos produced by these infected hens. However, in another study conducted by Landman et al. (58) eggs inoculated by dipping eggs into E. faecalis containing broth, or inoculating eggs via the air chambers of the egg cell, resulted in no disease condition. However, 6-day-old embryos inoculated with E. faecalis by the yolk sac route caused embryonic death, whereas albumin-inoculated eggs resulted in one of six birds developing arthritis. One-day-old chicks receiving E. faecalis by oral inoculation showed no signs of disease. Egg transmission of E. faecalis was also documented by a study conducted by Fertner et al. (59) in which newly hatched layer chicks were sampled from two hatches of Lohmann eggs (one from White parents and one from Brown parents). The hatchlings were sampled for E. faecalis at 0 hr and 24 hr following hatch. It was found that the prevalence of E. faecalis–infected birds increased from 14% (0 hr) to 97% (24 hr) in those hatchlings from the Brown parents and from 0.5% (0 hr) to 23% (24 hr) in the hatchlings from White parents. It was concluded that vertical transmission had occurred and that a few E. faecalis–contaminated embryos or eggs enable the rapid spread of the bacterium to other hatchlings. In a similar study in which nonviable embryos were evaluated from broiler hatcheries in western Canada, it was found that bacteria were isolated from 65.82% of dead embryos, with Enterococcus species being the most predominant isolates (29.71%) followed by E. coli (19.46%) (60). Of the Enterococcus species isolated, E. faecalis accounted for 79.58%. This report also demonstrated the use of matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) for speciating the isolated bacteria.

Based on the reports cited above, there is no doubt that vertical transmission of E. faecalis occurs. Here we offer three plausible mechanisms of how vertical transmission may happen. First is through eggshell contamination and penetration when fecal material (or other organic material from the environment) is present on the surface of the egg. This route of transmission may play an important role in infecting newly hatched chicks. Enterococcus faecalis grows well in egg yolks but not in egg whites (Reynolds, unpub. data). It has also been demonstrated that when E. faecalis was deposited on the eggshell membrane (along with other bacteria) it was not isolated from the albumen following incubation (61). Therefore, E. faecalis eggshell contamination may play a role in the late stages of embryonation when the embryo is pipping through the eggshell or being exposed to contaminated eggshells after hatching. Another potential mechanism is transovarial transmission by E. faecalis infecting the yolk through an infected hen. This would require the hen to become bacteremic, which could occur as a result of E. faecalis transcending the intestinal epithelium, entering the lymphovascular circulation (see above), and entering the yolk. Or initial infection could occur by a penetrating wound or some other integumentary or mucosal insult to the bird. A third mechanism by which E. faecalis may be entering the yolks or may be contributing to infertility is bacteriospermia. Bacteriospermia is a clinical syndrome in which bacteria colonize the male reproductive tract and infect the sperm, which leads to infertility. Bacteriospermia has been reported in humans and various animals including turkeys (62,63,64). Enterococcus faecalis has been identified in human cases of bacteriospermia (62,64).

Laying chickens

A condition termed amyloid arthropathy was first reported in heavy breed laying chickens by Landman et al. in 1994 (65). This initial preliminary report described a disease occurring in 5- to 6-wk-old and older birds with low morbidity. Affected birds were smaller and had an altered gait and swollen hocks. Necropsy findings revealed bronze-colored livers and orange-colored joint fluid. Histological results revealed amyloidosis. Infectious agents identified included S. faecalis and a reovirus. Subsequent to this initial report Landman et al. published a study whereby they described an animal model for reproducing articular amyloidosis by intravenously injecting laying pullets with high doses of E. faecalis (66). In a 2-yr study of amyloid arthropathy field cases in chickens Landman et al. reported several bacterial species were isolated. However, E. faecalis was identified as the primary causative agent, but not all E. faecalis isolates were capable of inducing the disease (67). It was also demonstrated that young birds exposed to aerosolized E. faecalis resulted in bacteremia, but no joint disease was induced. However, when birds were inoculated with high doses of E. faecalis by the intratracheal route, 30% of birds developed bacteremia and arthritic lesions (68). Birds that were inoculated by intramuscular injection via the pectoral or gastrocnemius muscles developed bacteremia and arthritis at a much higher incidence (100% and 90%, respectively) (68). In another European study, Petersen et al. demonstrated that some E. faecalis isolates obtained from chickens with amyloid arthropathy had colonial morphology differences (i.e., smaller, pinpoint colonies) (69). These smaller variant colonies were found to be more virulent than E. faecalis isolates having a larger, more typical, colonial morphology. In another study conducted by Petersen et al. 21 strains of E. faecalis were evaluated by Multilocus sequence typing, with 15 of the 21 having a sequence type of ST82, which indicated a wide geographic distribution of this sequence type (70). Although this study used E. faecalis isolates predominantly from Europe, it also included an isolate from the United States, indicating a global geographical distribution of the ST82 sequence type. More recent reports corroborated these findings indicating that E. faecalis was isolated from laying chickens experiencing stunted growth, lameness, and amyloid arthrosis in Canada (71), and sequence types ST82 and ST49 were identified in layer pullets displaying similar clinical disease (72). Interestingly, although amyloid arthropathy has been reported in brown layer breeds and less frequently in broiler breeders, it has not been reported in broiler chicks or white layer breeds that appear to be somewhat resistant (73). The Multilocus sequence types of ST36, ST59, ST89, ST170, ST171, ST172, and ST174 have been associated with amyloid arthropathy in layers (73). Sequence types ST32, ST176, ST177, and ST249 have been associated with amyloid arthropathy in broiler breeders (73,74).

Broiler chickens

Polyarticular amyloidosis was demonstrated in broiler breeders and was associated with an E. faecalis isolate (75). By employing pulsed-field gel electrophoresis it was demonstrated that the E. faecalis isolate belonged to the same clone as ones isolated previously from laying chickens including an isolate from brown layers in the United States (75). Enterococcus faecalis was shown to cause pulmonary hypertension in broiler chickens when high doses were administered either intravenously or intra-abdominally to birds (76). The E. faecalis isolate used was obtained from a field case, but no description of the case was provided. The authors suggested that the study provided a model for potential study of pulmonary hypertension in humans. A Danish group reported a case of septicemia and endocarditis in broiler breeders where a mixture of Streptococcus species and E. faecalis were isolated (48). The authors related that E. faecalis was responsible for septicemia in some birds and increased the overall flock mortality. A Canadian group reported on a study of broiler chicken farms in which several Enterococcus species were isolated (77). The vast majority of the Enterococcus species isolated were E. faecium with E. faecalis representing only 10%. Enterococcus faecalis isolates were found to have multiple-antibiotic resistance phenotypes and contained nine of the 12 virulence genes. A study conducted by Gregersen et al. examined 69 E. faecalis isolates originating from eight broiler breeder flocks demonstrating various types of lesions along with isolates from 20 birds without lesions from an additional two breeder flocks (74). The E. faecalis isolates were differentiated by Multilocus sequence typing. Twelve different sequence types were identified in which three sequence types made up 81% of the isolates. No correlations could be made between sequence type and lesion type between the diseased birds, and there was no correlation between sequence types in the healthy birds. Twelve E. faecalis strains were evaluated for antimicrobial resistance and sequence types. Six previously described sequence types along with two new sequence types were reported, with ST49 being the most frequently detected. However, no phylogenetic relationship could be identified. Although E. cecorum has been reported as the most frequent cause of vertebral osteomyelitis in broilers (78), E. faecalis has also been reported as causing vertebral osteomyelitis in broilers (79,80). Enterococcus faecalis has been reported in fresh feces of broiler chickens but rarely in the broiler litter (81). It was concluded that broiler litter selects against E. faecalis, and that broiler litter would be an unlikely environmental source of the bacterium.

Stepien-Pysniak et al. reported Dutch and Polish E. faecalis isolates originating from broiler chickens with yolk sac infections that were evaluated for virulence factors (15). Biochemical, MALDI-TOF MS, and rpoA gene sequencing techniques were used to identify isolates. A technique referred to as enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) along with MALDI-TOF MS has been used to characterize virulence factors and clonal relationships (15,82). In the report by Stepien-Pysniak et al. (15) of the 76 isolates identified as E. faecalis none demonstrated beta-hemolysis on blood agar; however, several isolates carried virulence genes. The ERIC-PCR results indicated about half of the isolates were genetically related. The MALDI-TOF MS cluster analysis data revealed relatedness that correlated to geographical origins of the isolates.

Turkeys

One of the earliest reports of E. faecalis in turkeys described liver granulomas associated with a S. faecalis bacterium (83). The authors observed that when the integrity of the intestinal epithelium was compromised, S. faecalis bacteria could be isolated from liver granulomas. They reported this to occur in field cases of hemorrhagic enteritis and when poults were orally inoculated with 24-hr broth cultures of S. faecalis. In a more recent study conducted on meat turkeys in Germany, a total of 28 Enterococcus species isolates were obtained from diagnostic cases of poults and young adult commercial meat turkeys (84). Enterococcus faecalis made up 10 isolates as did E. faecium, and eight isolates were E. gallinarum. These isolates were assessed for the genes attributed to five different virulence traits and were also evaluated for their virulence in a chicken embryo lethality assay. It was found that all E. faecalis isolates contained at least one virulence gene. The results of the chicken embryo lethality assay were variable. This variability was observed when testing the same isolate numerous times and between different isolates with some isolates causing high embryo mortality and other isolates causing only moderate to low mortality. There was no correlation established between genotype and phenotype; that is, a prediction of virulence could not be established based on the virulence genes detected. The authors concluded that “the lack of phenotypic expression despite genetic evidence indicates the presence of variant (‘loss-of-function‘) or silent genes that can be activated under in vivo conditions.”

Methods for assessing E. faecalis isolates

Isolation of E. faecalis is routinely done under aerobic culture conditions whereby samples are streaked onto sheep blood agar plates and Columbia colistin nalidixic acid agar (CNA agar, selective media for gram-positive cocci) and incubated for 48 hr. Typical colonies are selected and streaked again onto sheep blood agar for 24 hr to ensure purity of the selected colonies. Traditional methods of speciating E. faecalis include Gram staining followed by biochemical tests. This method is sensitive and relatively inexpensive but time consuming. The method of 16S rRNA and 18S rRNA gene sequencing is regarded as the gold standard for definitive identification (85). A more recent technology that is gaining wider usage is matrix-assisted laser desorption/ionization–time of flight mass spectroscopy (MALDI-TOF MS). MALDI-TOF MS allows the bacterium in question to be speciated within minutes following initial isolation and is highly accurate and now being used routinely in both medical and veterinary diagnostic laboratories (86,87,88,89). Stepien-Psyniak et al. (90) validated using MALDI-TOF MS to speciate Enterococcus species in wild birds. Of the 54 isolates tested all (100%) were identified by MALDI-TOF MS as Enterococcus species, which were confirmed by gene sequencing. It was found that 51 of 54 (94.4%) were identified to the species level and demonstrated that E. faecalis was the most prevalent species.

Chicken embryos have been used for decades to determine differences in virulence among bacterial strains (91,92,93,94). Typically, chicken embryos are inoculated with bacteria by the allantoic/chorioallantoic route and assessed for mortality at a predetermined time interval following inoculation. The embryo lethality assay (ELA) method has been used for a number of poultry pathogens including E. coli (95,96), Salmonella species (97), E. cecorum (98), and E. faecalis (99). In a very meticulous study, Blanco et al. established an LD50 of 6.6 colony-forming units/ml using an amyloid arthropathy strain of E. faecalis. This elaborate and statistically sound study was repeated four times and required 3443 egg embryos (99). The use of ELAs may yield useful information, but they are resource intensive and therefore are not ideal in a diagnostic laboratory setting. Furthermore, the correlation between genotype, virulence, and ELA results have been reported to be reliable for E. cecorum (98), but others have reported variable results when using ELAs to assess other bacteria, including E. coli and E. faecalis (84,94,95). Other methods have been developed for assessing virulence traits of E. faecalis isolates, including using microarray assays, PCRs for detecting virulence genes (15,39,100,101,102,103), and Multi Locus sequence typing (73,82). ERIC-PCR (see above) results did not correlate with ELA results (82). The use of MALDI-TOF MS to identify E. faecalis virulent strains that correlated with ELA virulence and geographical origin has been reported (15,104,105) and may prove to be a less resource intensive and more expedient method for determining virulence of isolates. However, to date no method has been reported to identify a definitive biomarker for virulence. A recent publication regarding E. faecalis infections in humans states “Despite the differentiation of commensal, clinical, and probiotic strains, there is no established correlation between the pathogenic potential or the presence of virulence genes and the origin of the strains” (23).

Enterococcus faecalis is a ubiquitous microbe occurring in the environment and in the intestinal tract of poultry. It often occurs in hatching eggs and the hatchery environment, where it can rapidly spread to hatchlings. Enterococcus faecalis is vertically transmitted either on or through the egg. A plausible mechanism in which eggs are infected is by transovarial transmission whereby E. faecalis gains access to the blood stream, perhaps by transcending the intestinal tract or by some other means. Enterococcus faecalis may be responsible for egg infertility and/or decreased hatchability. Enterococcus faecalis can cause amyloid arthropathy in older birds, and bacteremia may play an important role in this disease condition. Enterococcus faecalis has numerous virulence traits including antimicrobial resistance genes that are transferrable to other bacteria and formation of biofilms and the exotoxin cytolysin/hemolysin. How these virulence traits produce disease is not well understood. Methods to identify biomarkers for determining virulent versus avirulent E. faecalis strains have yet to be definitively established.

Abbreviations:

Abbreviations:
CNA =

Columbia colistin nalidixic acid agar;

ELA =

embryo lethality assay;

ERIC-PCR =

enterobacterial repetitive intergenic consensus polymerase chain reaction;

LD50 =

lethal dose 50%;

MALDI-TOF MS =

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

rRNA =

ribosomal ribonucleic acid;

ST =

sequence type

1.
Švec
P
,
Devriese
LA.
Enterococcus. In:
Bergey’s Manual of Systematics of Archaea and Bacteria
. p.
1
25
.
Published by John Wiley & Sons, Inc., in association with Bergey's Manual Trust
.
Hoboken, NJ
.
2015
.
2.
García-Solache
M
,
Rice
LB.
The enterococcus: a model of adaptability to its environment
.
Clin Microbiol Rev
.
32
;
2019
. .
3.
Fisher
K
,
Phillips
C.
The ecology, epidemiology and virulence of enterococcus
.
Microbiology
155
:
1749
1757
;
2009
.
4.
Schleifer
KH
,
Kilpper-Bälz
R.
Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov
.
Int J Syst Evol Microbiol
.
34
:
31
34
;
1984
.
5.
Lebreton
F
,
Manson
AL
,
Saavedra
JT
,
Straub
TJ
,
Earl
AM
,
Gilmore
MS.
Tracing the enterococci from paleozoic origins to the hospital
.
Cell
169
:
849
861
;
2017
.
6.
Fiore
E
,
Van Tyne
D
,
Gilmore
MS.
Pathogenicity of enterococci
.
Microbiol Spectr
.
7
:
1
23
;
2019
.
7.
Kayaoglu
G
,
Ørstavik
D.
Virulence factors of Enterococcus faecalis: relationship to endodontic disease
.
Crit Rev Oral Biol Med
.
15
:
308
320
;
2004
.
8.
Shah
D
,
Varahan
S.
Enterococcus faecalis
.
Trends Microbiol
.
32
(
9
):
925
926
;
2024
.
9.
Gilmore
MS
,
Lebreton
F
,
van Schaik
W.
Genomic transition of enterococci from gut commensals to leading causes of multidrug-resistant hospital infection in the antibiotic era
.
Curr Opin Microbiol
.
16
:
10
16
;
2013
.
10.
Coburn
PS
,
Gilmore
MS.
The Enterococcus faecalis cytolysin: a novel toxin active against eukaryotic and prokaryotic cells
.
Cell Microbiol
.
5
:
661
669
;
2003
.
11.
Furumura
MT
,
Figueiredo
PMS
,
Carbonell
GV
,
Darini
ALD
,
Yano
T.
Virulence-associated characteristics of strains isolated from clinical sources
.
Braz J Microbiol
.
37
:
230
236
;
2006
.
12.
Madsen
KT
,
Skov
MN
,
Gill
S
,
Kemp
M.
Virulence Factors Associated with Enterococcus faecalis infective endocarditis: a mini review
.
Open Microbiol J
.
11
:
1
11
;
2017
.
13.
Murase
K.
Cytolysin A (ClyA): a bacterial virulence factor with potential applications in nanopore technology, vaccine development, and tumor therapy
.
Toxins (Basel)
14
;
2022
. .
14.
Geraldes
C
,
Tavares
L
,
Gil
S
,
Oliveira
M.
Enterococcus virulence and resistant traits associated with its permanence in the hospital environment
.
Antibiotics (Basel)
11
(
7
):
857
,
2022
. .
15.
Stępień-Pyśniak
D
,
Hauschild
T
,
Dec
M
,
Marek
A
,
Urban-Chmiel
R
,
Kosikowska
U.
Phenotypic and genotypic characterization of Enterococcus spp. from yolk sac infections in broiler chicks with a focus on virulence factors
.
Poult Sci
.
100
:
100985
;
2021
.
16.
Van Tyne
D
,
Martin
MJ
,
Gilmore
MS.
Structure, function, and biology of the Enterococcus faecalis cytolysin
.
Toxins
5
:
895
911
;
2013
.
17.
Eaton
TJ
,
Gasson
MJ.
Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates
.
Appl Environ Microbiol
.
67
:
1628
1635
;
2001
.
18.
Franz
CM
,
Holzapfel
WH
,
Stiles
ME.
Enterococci at the crossroads of food safety
?
Int J Food Microbiol
.
47
:
1
24
;
1999
.
19.
Wells
CL
,
Jechorek
RP
,
Erlandsen
SL.
Evidence for the translocation of Enterococcus faecalis across the mouse intestinal tract
.
J Infect Dis
.
162
:
82
90
;
1990
.
20.
Wells
CL
,
Erlandsen
SL.
Localization of translocating Escherichia coli, Proteus mirabilis, and Enterococcus faecalis within cecal and colonic tissues of monoassociated mice
.
Infect Immun
.
59
:
4693
4697
;
1991
.
21.
Hrncir
T.
Gut microbiota dysbiosis: triggers, consequences, diagnostic and therapeutic options
.
Microorganisms
10
(
3
):
578
;
2022
. .
22.
Archambaud
C
,
Derré-Bobillot
A
,
Lapaque
N
,
Rigottier-Gois
L
,
Serror
P.
Intestinal translocation of enterococci requires a threshold level of enterococcal overgrowth in the lumen
.
Sci Rep
.
9
:
8926
;
2019
.
23.
Archambaud
C
,
Nunez
N
,
da Silva Ronni
AG
,
Kline Kimberly
A
,
Serror
P.
Enterococcus faecalis: an overlooked cell invader
.
Microbiol Mol Biol Rev
.
88
:
e00069–24
;
2024
.
24.
Nunez
N
,
Derré-Bobillot
A
,
Trainel
N
,
Lakisic
G
,
Lecomte
A
,
Mercier-Nomé
F
,
Cassard
A-M
,
Bierne
H
,
Serror
P
,
Archambaud
C.
The unforeseen intracellular lifestyle of Enterococcus faecalis in hepatocytes
.
Gut Microbes
14
:
2058851
;
2022
.
25.
da Silva
RAG
,
Tay
WH
,
Ho
FK
,
Tanoto
FR
,
Chong
KKL
,
Choo
PY
,
Ludwig
A
,
Kline
KA.
Enterococcus faecalis alters endo-lysosomal trafficking to replicate and persist within mammalian cells
.
PLoS Pathog
.
18
:
e1010434
;
2022
.
26.
Maekawa
S
,
Yoshioka
M
,
Kumamoto
Y.
Proposal of a new scheme for the serological typing of Enterococcus faecalis strains
.
Microbiol Immunol
.
36
:
671
681
;
1992
.
27.
Hufnagel
M
,
Hancock
LE
,
Koch
S
,
Theilacker
C
,
Gilmore
MS
,
Huebner
J.
Serological and genetic diversity of capsular polysaccharides in Enterococcus faecalis
.
J Clin Microbiol
.
42
:
2548
2557
;
2004
.
28.
Patti
JM
,
Höök
M.
Microbial adhesins recognizing extracellular matrix macromolecules
.
Curr Opin Cell Biol
.
6
:
752
758
;
1994
.
29.
Jett
BD
,
Jensen
HG
,
Nordquist
RE
,
Gilmore
MS.
Contribution of the pAD1-encoded cytolysin to the severity of experimental Enterococcus faecalis endophthalmitis
.
Infect Immun
.
60
:
2445
2452
;
1992
.
30.
Gaspar
FB
,
Crespo
MTB
,
Lopes
MFS.
Proposal for a reliable enterococcal cytolysin production assay avoiding apparent incongruence between phenotype and genotype
.
J Med Microbiol
.
58
:
1122
1124
;
2009
.
31.
Miyazaki
S
,
Ohno
A
,
Kobayashi
I
,
Uji
T
,
Yamaguchi
K
,
Goto
S.
Cytotoxic effect of hemolytic culture supernatant from Enterococcus faecalis on mouse polymorphonuclear neutrophils and macrophages
.
Microbiol Immunol
.
37
:
265
270
;
1993
.
32.
Libertin
CR
,
Dumitru
R
,
Stein
DS.
The hemolysin/bacteriocin produced by enterococci is a marker of pathogenicity
.
Diagn Microbiol Infect Dis
.
15
:
115
120
;
1992
.
33.
Reynolds
DL
,
Simpson
EB
,
Hille
MM
.
A method for demonstrating the cytolysin/hemolysin of enterococcus faecalis isolates of poultry origin
.
Poultry
.
4
:
11
;
2025
.
34.
Poeta
P
,
Costa
D
,
Klibi
N
,
Rodrigues
J
,
Torres
C.
Phenotypic and genotypic study of gelatinase and beta-haemolysis activities in faecal enterococci of poultry in Portugal
.
J Vet Med B Infect Dis Vet Public Health
53
:
203
208
;
2006
.
35.
Hughes
ER
,
Winter
SE.
Enterococcus faecalis: E. coli’s siderophore-inducing sidekick
.
Cell Microbe
20
:
411
412
;
2016
.
36.
Clewell
DB
,
Weaver
KE.
Sex pheromones and plasmid transfer in Enterococcus faecalis
.
Plasmid
21
:
175
184
;
1989
.
37.
Sterling
AJ
,
Snelling
WJ
,
Naughton
PJ
,
Ternan
NG
,
Dooley
JSG.
Competent but complex communication: The phenomena of pheromone-responsive plasmids
.
PLoS Pathog
.
16
:
e1008310
;
2020
.
38.
Manson
AL
,
Van Tyne
D
,
Straub
TJ
,
Clock
S
,
Crupain
M
,
Rangan
U
,
Gilmore
MS
,
Earl
AM.
chicken meat-associated enterococci: influence of agricultural antibiotic use and connection to the clinic
.
Appl Environ Microbiol
.
85
(
22
);
2019
. .
39.
Olsen
RH
,
Schønheyder
HC
,
Christensen
H
,
Bisgaard
M.
Enterococcus faecalis of human and poultry origin share virulence genes supporting the zoonotic potential of E. faecalis
.
Zoonoses Public Health
59
:
256
263
;
2012
.
40.
Poulsen
LL
,
Bisgaard
M
,
Son
NT
,
Trung
NV
,
An
HM
,
Dalsgaard
A.
Enterococcus faecalis clones in poultry and in humans with urinary tract infections, Vietnam
.
Emerg Infect Dis
.
18
:
1096
1100
;
2012
.
41.
Jha
R
,
Das
R
,
Oak
S
,
Mishra
P.
Probiotics (direct-fed microbials) in poultry nutrition and their effects on nutrient utilization, growth and laying performance, and gut health: a systematic review
.
Animals (Basel)
.
10
(
10
):
1863
;
2020
. .
42.
Mountzouris
KC
,
Tsitrsikos
P
,
Palamidi
I
,
Arvaniti
A
,
Mohnl
M
,
Schatzmayr
G
,
Fegeros
K.
Effects of probiotic inclusion levels in broiler nutrition on growth performance, nutrient digestibility, plasma immunoglobulins, and cecal microflora composition
.
Poult Sci
.
89
:
58
67
;
2010
.
43.
Rathnayake
M
,
Ranaraja
A
,
Popowich
S
,
Gautam
H
,
Subhasinghe
I
,
Ahmed
KA
,
Chow-Lockerbie
B
,
Ayalew
L
,
Gomis
S.
Colonization of probiotic bacteria in the intestine of chicken embryos following coarse spray on incubating eggs. In:
Proceedings of the Sixty-Seventh Annual Meeting of the American Association of Avian Pathologists 2024
,
St. Louis, MO
. July 9–11;
American Association of Avian Pathologists, Jacksonville, FL
.; p.
85
;
2024
.https://aaap.memberclicks.net/assets/2024_Annual_Meeting/AAAP%202024%20Proceedings%20Draft%201.pdf.
44.
Shehata
AA
,
Tarabees
R
,
Basiouni
S
,
ElSayed
MS
,
Gaballah
A
,
Krueger
M.
Effect of a potential probiotic candidate Enterococcus faecalis-1 on growth performance, intestinal microbiota, and immune response of commercial broiler chickens
.
Probiotics Antimicrob Proteins
12
:
451
460
;
2020
.
45.
Wang
X
,
Yang
Y
,
Huycke
MM.
Risks associated with enterococci as probiotics
.
Food Res Int
.
129
:
108788
;
2020
.
46.
Walker
GK
,
Suyemoto
MM
,
Gall
S
,
Chen
L
,
Thakur
S
,
Borst
LB.
The role of Enterococcus faecalis during co-infection with avian pathogenic Escherichia coli in avian colibacillosis
.
Avian Pathol
.
49
:
589
599
;
2020
.
47.
Karunarathna
R
,
Ahmed
KA
,
Goonewardene
K
,
Gunawardana
T
,
Kurukulasuriya
S
,
Liu
M
,
Gupta
A
,
Popowich
S
,
Ayalew
L
,
Chow-Lockerbie
B
,et al.
Exposure of embryonating eggs to Enterococcus faecalis and Escherichia coli potentiates E. coli pathogenicity and increases mortality of neonatal chickens
.
Poult Sci
.
101
:
101983
;
2022
.
48.
Chadfield
MS
,
Christensen
JP
,
Christensen
H
,
Bisgaard
M.
Characterization of streptococci and enterococci associated with septicaemia in broiler parents with a high prevalence of endocarditis
.
Avian Pathol
.
33
:
610
617
;
2004
.
49.
Eyssen
H
,
Desomer
P.
Effects of Streptococcus faecalis and a filterable agent on growth and nutrient absorption in gnotobiotic chicks
.
Poult Sci
.
46
:
323
333
;
1967
. .
50.
Devriese
LA
,
Hommez
J
,
Wijfels
R
,
Haesebrouck
F.
Composition of the enterococcal and streptococcal intestinal flora of poultry
.
J Appl Bacteriol
.
71
:
46
50
;
1991
.
51.
Dolka
B
,
Gołębiewska-Kosakowska
M
,
Krajewski
K
,
Kwieciński
P
,
Nowak
T
,
Szubstarski
J
,
Wilczyński
J
,
Szeleszczuk
P.
Occurrence of Enterococcus spp. in poultry in Poland based on 2014–2015 data
.
Medycyna weterynaryjna
73
(
4
):
220
224
;
2017
. .
52.
Olsen
RH
,
Frantzen
C
,
Christensen
H
,
Bisgaard
M.
An investigation on first-week mortality in layers
.
Avian Dis
.
56
:
51
57
;
2012
.
53.
Alaboudi
AR
,
Hammed
DA
,
Basher
HA
,
Hassan
MG.
Potential pathogenic bacteria from dead in shell chicken embryos
.
Iraqi J Vet Sci
.
5
:
109
114
;
1992
.
54.
Reynolds
DL
,
Akinc
S.
Decreased hatchability (fertility) associated with a Streptococcus species.
Oral presentation at the North Central Avian Disease Conference
. October 3–5,
Columbus, OH
.
1993
.
55.
Reynolds
DL
,
Loy
JD.
Decrease in hatchability of pheasant eggs associated with Enterococcus faecalis
.
Avian Dis
.
64
:
517
521
;
2020
.
56.
Tankson
JD
,
Thaxton
JP
,
Vizzier‐Thaxton
Y.
Bacteria in heart and lungs of young chicks
.
J Appl Microbiol
.
92
:
443
450
;
2002
.
57.
Landman
WJ
,
Feberwee
A
,
Mekkes
DR
,
Veldman
KT
,
Mevius
DJ.
A study on the vertical transmission of arthropathic and amyloidogenic Enterococcus faecalis
.
Avian Pathol
.
28
:
559
566
;
1999
.
58.
Landman
WJ
,
Mekkes
DR
,
Chamanza
R
,
Doornenbal
P
,
Gruys
E.
Arthropathic and amyloidogenic Enterococcus faecalis infections in brown layers: a study on infection routes
.
Avian Pathol
.
28
:
545
557
;
1999
.
59.
Fertner
M
,
Olsen
R
,
Bisgaard
M
,
Christensen
H.
Transmission and genetic diversity of Enterococcus faecalis among layer chickens during hatch
.
Acta Vet Scand
.
53
:
56
;
2011
.
60.
Karunarathna
R
,
Popowich
S
,
Wawryk
M
,
Chow-Lockerbie
B
,
Ahmed
KA
,
Yu
C
,
Liu
M
,
Goonewardene
K
,
Gunawardana
T
,
Kurukulasuriya
S
,et al.
Increased incidence of enterococcal infection in nonviable broiler chicken embryos in western Canadian hatcheries as detected by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry
.
Avian Dis
.
61
:
472
480
;
2017
.
61.
Dolman
J
,
Board
RG.
The influence of temperature on the behaviour of mixed bacterial contamination of the shell membrane of the hen’s egg
.
Epidemiol Infect
.
108
:
115
21
;
1992
.
62.
Hamazah
LM
,
Al-Dahmoshi
HOM.
Molecular investigation of cytolysin genes among bacterial isolates recovered from pyospermic patients in Hilla City, Iraq
.
J Appl Nat Sci
.
14
:
1110
1118
;
2022
.
63.
Lenický
M
,
Slanina
T
,
Kačániová
M
,
Galovičová
L
,
Petrovičová
M
,
Ďuračka
M
,
Benko
F
,
Kováč
J
,
Tvrdá
E.
Identification of bacterial profiles and their interactions with selected quality, oxidative, and immunological parameters of turkey semen
.
Animals (Basel)
11
(
6
):
1771
;
2021
. .
64.
Tvrdá
E
,
Ďuračka
M
,
Benko
F
,
Lukáč
N.
Bacteriospermia—a formidable player in male subfertility
.
Open Life Sci
.
17
:
1001
1029
;
2022
.
65.
Landman
WJM
,
Gruys
E
,
Dwars
RM.
A syndrome-associated with growth depression and amyloid arthropathy in layers—a preliminary report
.
Avian Pathol
.
23
:
461
470
;
1994
.
66.
Landman
WJM
,
Peperkamp
NHMT
,
Koch
CAM
,
Tooten
PCJ
,
Crauwels
PAP
,
Gruys
E.
Induction of amyloid arthropathy in chickens
.
Amyloid
4
:
87
97
;
1997
.
67.
Landman
WJ
,
vd Bogaard
AE
,
Doornenbal
P
,
Tooten
PC
,
Elbers
AR
,
Gruys
E.
The role of various agents in chicken amyloid arthropathy
.
Amyloid
5
:
266
278
;
1998
.
68.
Landman
WJ
,
Veldman
KT
,
Mevius
DJ
,
van Eck
JH.
Investigations of Enterococcus faecalis–induced bacteraemia in brown layer pullets through different inoculation routes in relation to the production of arthritis
.
Avian Pathol
.
32
:
463
471
;
2003
.
69.
Petersen
A
,
Chadfield
MS
,
Christensen
JP
,
Christensen
H
,
Bisgaard
M.
Characterization of small-colony variants of Enterococcus faecalis isolated from chickens with amyloid arthropathy
.
J Clin Microbiol
.
46
:
2686
2691
;
2008
.
70.
Petersen
A
,
Christensen
H
,
Philipp
HC
,
Bisgaard
M.
Clonality of Enterococcus faecalis associated with amyloid arthropathy in chickens evaluated by multilocus sequence typing (MLST)
.
Vet Microbiol
.
134
:
392
395
;
2009
.
71.
Petrik
M.
Enterococcus fecalis infections in laying hens. In:
Proceedings of the Sixty-Seventh Annual Meeting of the American Association of Avian Pathologists 2024
,
St. Louis, MO
. July 9–11;
American Association of Avian Pathologists, Jacksonville, FL
.; p.
18
;
2024
. https://aaap.memberclicks.net/assets/2024_Annual_Meeting/AAAP%202024%20Proceedings%20Draft%201.pdf.
72.
Gore
A
,
Crespo
R
,
Walker
G
,
Suyemoto
M
,
Petric
M.
Whole genome sequencing and characterization of Enterococcus faecalis isolated from pullet layers with growth depression and amyloid arthropathy. In:
Proceedings of the Sixty-Seventh Annual Meeting of the American Association of Avian Pathologists 2024
,
St. Louis, MO
. July 9–11;
American Association of Avian Pathologists, Jacksonville, FL
.; p.
71
;
2024
. https://aaap.memberclicks.net/assets/2024_Annual_Meeting/AAAP%202024%20Proceedings%20Draft%201.pdf.
73.
Blanco
AE
,
Barz
M
,
Icken
W
,
Cavero
D
,
Mazaheri
A
,
Voss
M
,
Schmutz
M
,
Preisinger
R.
Twenty years of amyloid arthropathy research in chickens
.
World’s Poult Sci J
.
72
:
495
508
;
2016
.
74.
Gregersen
RH
,
Petersen
A
,
Christensen
H
,
Bisgaard
M.
Multilocus sequence typing of Enterococcus faecalis isolates demonstrating different lesion types in broiler breeders
.
Avian Pathol
.
39
:
435
440
;
2010
.
75.
Steentjes
A
,
Veldman
KT
,
Mevius
DJ
,
Landman
WJ.
Molecular epidemiology of unilateral amyloid arthropathy in broiler breeders associated with Enterococcus faecalis
.
Avian Pathol
.
31
:
31
39
;
2002
.
76.
Tankson
JD
,
Thaxton
JP
,
Vizzier-Thaxton
Y.
Pulmonary hypertension syndrome in broilers caused by Enterococcus faecalis
.
Infect Immun
.
69
:
6318
6322
;
2001
.
77.
Diarra
MS
,
Rempel
H
,
Champagne
J
,
Masson
L
,
Pritchard
J
,
Topp
E.
Distribution of antimicrobial resistance and virulence genes in Enterococcus spp. and characterization of isolates from broiler chickens
.
Appl Environ Microbiol
.
76
:
8033
8043
;
2010
.
78.
Jung
A
,
Chen
LR
,
Suyemoto
MM
,
Barnes
HJ
,
Borst
LB.
A Review of Enterococcus cecorum infection in poultry
.
Avian Dis
.
62
:
261
271
;
2018
.
79.
Braga
JFV
,
Leal
CAG
,
Silva
CC
,
Fernandes
AA
,
Martins
NRdS
,
Ecco
R.
Genetic diversity and antimicrobial resistance profile of Enterococcus faecalis isolated from broilers with vertebral osteomyelitis in Southeast Brazil
.
Avian Pathol
.
47
:
14
22
;
2018
.
80.
Menck-Costa
M
,
Huijboom
J
,
Souza
M
,
Justino
L
,
Bracarense
AP
,
Pereira
U
,
Baptista
A.
Vertebral osteomyelitis caused by Enterococcus faecalis in broiler chickens from southern Brazil
.
Pesq Vet Bras
.
44
;
2024
. .
81.
Kuntz
RL
,
Hartel
PG
,
Rodgers
K
,
Segars
WI.
Presence of Enterococcus faecalis in broiler litter and wild bird feces for bacterial source tracking
.
Water Res
.
38
:
3551
3557
;
2004
.
82.
Blanco
AE
,
Barz
M
,
Cavero
D
,
Icken
W
,
Sharifi
AR
,
Voss
M
,
Buxadé
C
,
Preisinger
R.
Characterization of Enterococcus faecalis isolates by chicken embryo lethality assay and ERIC-PCR
.
Avian Pathol
.
47
:
23
32
;
2018
.
83.
Moore
WEC
,
Gross
WB.
Liver Granulomas of turkeys: causative agents and mechanism of infection
.
Avian Dis
.
12
:
417
422
;
1968
.
84.
Maasjost
J
,
Lüschow
D
,
Kleine
A
,
Hafez
HM
,
Mühldorfer
K
,
Bondi
M.
Presence of virulence genes in Enterococcus species isolated from meat turkeys in Germany does not correlate with chicken embryo lethality
.
BioMed Res Int
.;
2019
. .
85.
Singhal
N
,
Kumar
M
,
Kanaujia
PK
,
Virdi
JS.
MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis
.
Front Microbiol
.
6
:
791
;
2015
.
86.
Ashfaq
MY
,
Da’na
DA
,
Al-Ghouti
MA.
Application of MALDI-TOF MS for identification of environmental bacteria: a review
.
J Environ Manage
.
305
:
114359
;
2022
.
87.
Calderaro
A
,
Chezzi
C.
MALDI-TOF MS: a reliable tool in the real life of the clinical microbiology laboratory
.
Microorganisms
12
:
322
;
2024
.
88.
Hou
TY
,
Chiang-Ni
C
,
Teng
SH.
Current status of MALDI-TOF mass spectrometry in clinical microbiology
.
J Food Drug Anal
.
27
:
404
414
;
2019
.
89.
Stępień-Pyśniak
D
,
Marek
A
,
Banach
T
,
Adaszek
Ł
,
Pyzik
E
,
Wilczyński
J
,
Winiarczyk
S.
Prevalence and antibiotic resistance of Enterococcus strains isolated from poultry
.
Acta Vet Hung
.
64
:
148
163
;
2016
.
90.
Stępień-Pyśniak
D
,
Hauschild
T
,
Różański
P
,
Marek
A.
MALDI-TOF mass spectrometry as a useful tool for identification of Enterococcus spp. from wild birds and differentiation of closely related species
.
J Microbiol Biotechnol
.
27
:
1128
1137
;
2017
.
91.
Finkelstein
RA
,
Ransom
JP.
Non-specific resistance to experimental cholera in embryonated eggs
.
J Exp Med
.
112
:
315
328
;
1960
.
92.
Goodpasture
EW.
Use of embryo chick in investigation of certain pathological problems originally published in Southern Medical Journal, May 1933
.
South Med J
.
76
:
553
555
;
1983
.
93.
Goodpasture
EW
,
Anderson
K.
The problem of infection as presented by bacterial invasion of the chorio-allantoic membrane of chick embryos
.
Am J Pathol
.
13
:
149
174
;
1937
.
94.
Powell
CJ,
Finkelstein
RA.
Virulence of Escherichia coli strains for chick embryos
.
J Bacteriol
.
91
:
1410
1417
;
1966
.
95.
Nolan
LK
,
Wooley
RE
,
Brown
J
,
Spears
KR
,
Dickerson
HW
,
Dekich
M.
Comparison of a complement resistance test, a chicken embryo lethality test, and the chicken lethality test for determining virulence of avian Escherichia coli
.
Avian Dis
.
36
:
395
397
;
1992
.
96.
Wooley
R
,
Gibbs
P
,
Brown
T
,
Maurer
J.
Chicken embryo lethality assay for determining the virulence of avian Escherichia coli isolates
.
Avian Dis
.
44
:
318
324
;
2000
.
97.
Zhang
JF
,
Wei
B
,
Cha
SY
,
Shang
K
,
Jang
HK
,
Kang
M.
The use of embryonic chicken eggs as an alternative model to evaluate the virulence of Salmonella enterica serovar Gallinarum
.
PLoS One
15
:
e0238630
;
2020
.
98.
Borst
LB
,
Suyemoto
MM
,
Keelara
S
,
Dunningan
SE
,
Guy
JS
,
Barnes
HJ.
A chicken embryo lethality assay for pathogenic Enterococcus cecorum
.
Avian Dis
.
58
:
244
248
;
2014
.
99.
Blanco
AE
,
Barz
M
,
Icken
W
,
Cavero
D
,
Sharifi
AR
,
Voss
M
,
Preisinger
R
,
Buxadé
C.
Chicken embryo lethality assay for determining the lethal dose and virulence of Enterococcus faecalis
.
Avian Pathol
.
46
:
548
555
;
2017
.
100.
Hwang
IY
,
Lim
SK
,
Ku
HO
,
Park
CK
,
Jung
SC
,
Park
YH
,
Nam
HM.
Occurrence of virulence determinants in fecal Enterococcus faecalis isolated from pigs and chickens in Korea
.
J Microbiol Biotechnol
.
21
:
1352
1355
;
2011
.
101.
Champagne
J
,
Diarra
MS
,
Rempel
H
,
Topp
E
,
Greer
CW
,
Harel
J
,
Masson
L.
Development of a DNA microarray for enterococcal species, virulence, and antibiotic resistance gene determinations among isolates from poultry
.
Appl Environ Microbiol
.
77
:
2625
2633
;
2011
.
102.
Poeta
P
,
Costa
D
,
Klibi
N
,
Rodrigues
J
,
Torres
C.
Phenotypic and genotypic study of gelatinase and beta-haemolysis activities in faecal enterococci of poultry in Portugal
.
J Vet Med B
53
:
203
208
;
2006
.
103.
Silva
N
,
Igrejas
G
,
Vaz
J
,
Araújo
C
,
Cardoso
L
,
Rodrigues
J
,
Torres
C
,
Poeta
P.
Virulence factors in enterococci from partridges (Alectoris rufa) representing a food safety problem
.
Foodborne Path Dis
.
8
:
831
833
;
2011
.
104.
Reynolds
DL
,
Simpson
EB
,
Hille
MM
,
Holz
EM
,
Jia
B.
Determining the virulence of Enterococcus faecalis isolates from commercial poultry
. In: Proceedings of the 75th North Central Avian Disease Conference; April 16–17;
Minneapolis, MN
.
Program Chair, Tim Johnson
; p.
20
;
2024
.
105.
Reynolds
DL
,
Hille
MM
,
Holz
EM
,
Simpson
EB.
Using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) for determining virulence of Enterococcus faecalis isolates of poultry origin
.
Int J Vet Sci. In press
.;
2025
. .