Enterococcus hirae–Associated Endocarditis Outbreak in Young Broiler Breeders of the Female Line Open Access

Enterococcal infections in poultry were reported as early as 1947 and were described as “fecal strep” infection. In those years, Enterococcus species were still assigned to the Streptococcus Lancefield antigenic serogroup D. In 1984, the genus named Enterococcus was formed; these are Gram-positive coccoid bacteria (1). Enterococcus spp. are important members of the intestinal microflora in poultry. Strains frequently isolated from the intestinal flora of chickens are Enterococcus faecium, Enterococcus faecalis, Enterococcus cecorum, Enterococcus hirae, and Enterococcus durans (2). All these Enterococcus spp. are associated with pathological changes in chickens (1), for example, amyloid arthropathy in layers (E. faecalis) or bacterial osteomyelitis of the free thoracic vertebra in broilers breeders (E. cecorum) (3,4).

Enterococci infections may result in either an acute or a subacute to chronic form. In the acute form, birds rarely present clinical signs prior to being found dead, whereas in the subacute or chronic form, clinical signs can be observed, such as retarded growth, lameness, head tremors, and depression. These signs depend on the pathological lesions (i.e., arthritis, osteomyelitis, pericarditis, and valvular endocarditis). In the case of endocarditis, vegetative lesions can be found on the atrioventricular (AV) valves of the heart, both on the left and right side (1). Bacterial endocarditis is usually caused by streptococci or enterococci. Among these are E. hirae, E. faecalis, Streptococcus zooepidemicus, and Streptococcus gallinaceus, which can be isolated from naturally occurring infections (5,6,7).

In experimental studies, bacterial endocarditis has been reproduced. Chadfield et al. (8) inoculated E. hirae in 3-wk-old broiler chickens intravenously. After only 5 days, the birds died and showed typical alterations on the AV valves. In naturally occurring infections in broilers, a peak in mortality is observed in the second week of life, suggesting that broiler chickens are already infected early in life with E. hirae (8). Endocarditis due to an infection of E. hirae was previously described in broiler breeders in the rearing period (6), but differences in the prevalence of endocarditis between chickens of the male and female line of broiler breeders have not been described. Difference in disease susceptibility is thought to be attributed to the levels of natural antibodies (NAb), as described previously (9). NAb are antibodies found in all vertebrates studied so far, including birds. These antibodies recognize an antigen without a previous exposure to that specific antigen. NAb likely reflect an early defense toward infections and may represent a clearing mechanism of endogenous dangerous material, such as necrotic or tumor cells (10). In chickens, layers bred for enhanced NAb levels binding keyhole limpet hemocyanin (KLH) were found to have a higher resistance to Escherichia coli infections than birds with low NAb levels binding KLH (11). It is extremely unlikely that chickens are exposed to KLH in their life; therefore, antibodies binding to KLH can be considered NAb (11).

In this case report, we describe the macroscopic and bacteriological findings of an E. hirae–associated endocarditis case. In addition, we determined the levels of NAbs in the affected flocks. Enterococcus hirae–associated endocarditis lesions were only observed in the female line of these broiler breeders.

On a rearing farm with a capacity of 46,500 animals, Ross 308 broiler breeders were kept in four separate houses. Birds from the male line were kept in House 1 (n = 4,500 in total); the 42,000 pullets of the female line were housed in the other three houses. All houses were connected by a central corridor. A strict biosecurity protocol is maintained (e.g., every visitor is required to shower prior to entry into the facilities). Once the facilities were entered, the internal biosecurity was not strictly implemented; the same equipment (e.g., boots, overalls, buckets) were used in all houses. Cleaning and disinfection protocol applied between production cycles was identical for all houses. Taken together, Houses 1–4 were considered one biosecurity and epidemiologic unit.

From 2016 to 2021, in every rearing period, starting at 2–3 wk posthatch (ph) and until 6-wk ph, increased mortality occurred. Peak of mortality (up to 0.52% per week) was at 4 wk ph and thereafter declined to acceptable levels of 0.15% per week. The attending veterinarian diagnosed the pullets with endocarditis caused by E. hirae, after performing gross pathology and bacteriology. This diagnosis was confirmed in consecutive rearing periods, and for that reason, the case was presented to the Veterinary Faculty of Utrecht University (the Netherlands).

One flock was followed during its rearing period for this case report. The cumulative mortality between 2 and 6 wk in the male line (House 1) was 319 birds (7.09%), and in the female line, the mortality was 106 (1.33%), 173 (1.02%), and 240 (1.41%) birds in Houses 2–4, respectively. The mortality in the male line included 5.65% caused by selection and culling by the farmer. Males were culled for two reasons: birds retarded in growth or birds with unilateral or bilateral lameness. Previously, in this rearing period, a Staphylococcus aureus–associated arthritis was diagnosed by the attending veterinarian by gross pathology and bacteriology. In preceding rearing periods, no extensive mortality caused by Staphylococcus aureus–associated arthritis was reported; therefore, the E. hirae endocarditis was further investigated in this case.

Necropsy was performed on six occasions between 12 and 47 days ph (dph). In total, 256 birds were examined. Mortality of the previous 3 days was collected per house and macroscopically examined. For the necropsy at 12 dph, only birds that died in the 24 hr prior were examined.

In House 1, a total of 65 male line birds were macroscopically examined. The most prominent findings were arthritis (n = 12), femoral head necrosis (n = 11), and polyserositis (n = 9). Several birds retarded in growth were culled by the farmer (n = 10). No clear diagnosis for the retarded growth could be determined, and no further additional diagnostic tests were performed. Of the 65 birds examined, no bird was diagnosed with endocarditis.

In total, 191 birds of the female line were examined, 39, 66, and 86 originated from Houses 2–4. From those, 189 were females, and 2 were males. The main cause of mortality appeared related to endocarditis (51.8%; Table 1). Endocarditis was found on the left, right, or on both AV valves; however, no lesions on the left AV valves were observed after 26 dph. Endocarditis on the right AV valve (Fig. 1A,B) was still found until 47 dph (Table 1). Enlarged hearts and dilation of the atria were observed in association with valvular endocarditis (Fig. 1A). Ascites was found from 26 dph onward, but only in three birds, despite the high prevalence of endocarditis on the right AV valve. Furthermore, embolic lesions were found in the aorta and in the arteria ischiadica externa starting at 18 dph (Fig. 1D,E), followed by the presence of emboli in the larger branches of the arteria pulmonalis at 30 dph (Fig. 1C). Hepatomegaly, occasionally hepatitis, and splenomegaly were observed in many birds with endocarditis. In 18 (9.4%) of all examined birds, the femur head broke after exarticulation of the femur from the hip joint. In addition, some of these birds showed enlarged livers. Both males of the female line (Houses 3 and 4) were diagnosed with endocarditis.

Other macroscopic findings, such as broken femurs, polyserositis, and slipped tendons, were found during gross pathology assessments. These findings were considered of minor impact, and the incidence (in total, <5% of the examined birds) was very low.

All samples were collected aseptically and cultured on Columbia Sheep Blood agar plates (Oxoid, Wesel, Germany) and incubated for 24 hr at 37°C in a 5% CO₂-enriched atmosphere. Colonies were identified by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry.

Samples from bone marrow (n = 4, 3, 1, and 3) from birds of the male line were collected at 18, 26, 33, and 40 dph, respectively, and submitted for bacteriological examination. In addition, one sample of a swollen hock joint was collected at 18 dph and submitted. From the bone marrow samples, four (36.4%) did not show bacterial growth, in six (54.5%), Staphylococcus aureus was detected, and in one (9.1%), Staphylococcus xylosus was found. Staphylococcus aureus was also detected in the hock joint sample. No E. hirae was detected in these samples.

The results of the bacteriological examination of the samples collected from the female line birds are outlined in Table 1. In brief, a total of 45 bone marrow samples were collected, from which 29 (64.4%) yielded E. hirae. In addition, two heart samples were cultured, and E. hirae was detected in both. On three occasions, Escherichia coli was cultured from the bone marrow.

Blood samples (n = 35, 34, and 30) were collected from Houses 1, 2, and 4, respectively. Serum was harvested and tested for the presence of NAb titers of the isotypes immunoglobulin (Ig) G and IgM. These were determined by using an indirect ELISA, as described by (12). In Table 2, the IgG and IgM titers of the KLH NAbs are shown. In brief, the means (±SD) of the IgG titers of the male line in House 1 were 3.47 (0.57) compared to 4.69 (0.76) and 4.77 (1.07) in Houses 2 and 4, respectively. The difference between the titers obtained in the male line (House 1) and the female line (Houses 2 and 4) was statistically significant. When IgM KLH NAb were assessed in the female and male lines, a higher level of IgM NAb was detected in House 1 (male) compared with House 2 (female; P < 0.05; Table 2).

The intervention strategies were focused on the breeder farm and on the breeding company. As E. hirae–associated endocarditis was diagnosed in several consecutive rearing periods, and Staphylococcus aureus–associated arthritis was an incidental diagnosis, the intervention strategies are solely focused on the prevention of E. hirae.

In this rearing period, cumulative mortality rates in the female line exceeded the breeder’s standards (13). However, no antibiotic treatment was administered for two reasons: the mortality rate was only slightly increased in the period of 2 to 6 wk ph (i.e., 1.02%–1.41%), and antibiotic treatment for endocarditis often fails (1,14). If antibiotic treatment is considered in acute or subacute infection, an antimicrobial susceptibility test should be performed, as most E. hirae isolates present multidrug-resistant phenotypes (15).

Although the clonality of the E. hirae strains involved in these outbreaks was never determined, the repeated problems on this farm suggest a farm-specific problem. Therefore, emphasis on the improvement of cleaning and disinfection strategies is key and was recommended between production cycles.

To our knowledge, no other broiler breeder rearing farms experienced outbreaks of E. hirae–associated endocarditis at the same time this outbreak occurred. Therefore, chickens were likely infected shortly after placement. Despite this, in Enterococcus outbreaks, the hygiene status of the hatchery should be taken into consideration as a potential source of contamination.

This case suggests a difference in susceptibility to E. hirae–associated infections in female and male lines. Genetic variation in susceptibility to colibacillosis has been previously observed (16,17), both suggesting that selection for reduced susceptibility is possible. Although the genetic effect of E. hirae susceptibility has not been studied, the difference in the incidence of endocarditis in the female and male lines suggests a potential effect. The study of the basis of this line’s susceptibility difference might help breed resistance to Enterococcus spp. infections in chicks in the future.

Endocarditis in broiler breeders can be associated with several streptococci and enterococci (e.g., Streptococcus gallinaceus, E. hirae) (5,6). In a previous case, sudden deaths over the first weeks of life of broiler breeders, with typical lesions as cardiac enlargement and endocarditis, were found, and E. hirae was cultured from these lesions (6). In the present report, similar clinical findings were found, even though the incidence of macroscopic findings changed over time. Endocarditis of the left AV valves was observed in birds that had died up to 26 dph, while endocarditis on the right AV valves occurred in birds that died up to 47 dph (Table 1). The persistence of these lesions in the right AV valve might be related with the anatomy of the chicken’s heart. Although the left AV valve is a poorly defined tricuspid valve, the right AV valve is a single flap of myocardium attached to the free wall of the right ventricle, being bilaminar only on its upper portion (18). These differences might end in blood stasis in the right AV valve inducing endocarditis more easily. In E. hirae–associated endocarditis outbreaks in broilers, similar distribution of lesions in the heart over time were reported (7). Emboli in the arteria ischiadica externa and branches of the arteria pulmonalis were observed (Fig. 1C–E). These emboli could have been caused by embolism from valvular vegetations or by hematogenous spreading of bacteria, followed by adhesion and development of vascular lesions from multiplying enterococci. For thromboembolism, lesions would be expected in the smaller branches of the arteries; therefore, it has been hypothesized that small aggregates of bacteria travel through the cardiovascular system and, after adhesion to the endothelium, cause vegetative lesions (7).

Most enterococci species are inhabitants of the gastrointestinal tract of chickens. Among those, E. hirae is a commensal in the small intestine of broilers and was detected in birds starting at 3 wk of age (2). Bacteremia is a prerequisite for E. hirae to adhere to the endocardium and cause vegetative lesions. Subsequently, E. hirae–associated endocarditis can be reproduced by intravenous inoculation of the bacteria (8). Three routes of translocation into the blood stream are possible: 1) translocation across the intestinal epithelium; 2) translocation from the respiratory tract; and 3) via skin lesions (1). No reports of translocation of enterococci across the intestinal epithelium in poultry have been published. Only translocations of E. faecalis across enterocytes and in the intestinal epithelium in vitro and in vivo (mouse model) have been reported (19,20). However, aerosol transmission of E. faecalis has been reported, resulting in acute septicemia in chickens (21). The exact route of infection in this specific case report remains unclear.

In E. hirae experimentally inoculated broilers, the disease onset is 5 to 16 days after inoculation (8). In the present case, endocarditis was first diagnosed at 12 dph, suggesting that the birds were infected in the first week of life, supporting the idea of E. hirae persisting in the farm. However, the clonality of the E. hirae strains involved was not determined. Other studies found both polyclonal and clonal infections by using pulsed-field gel electrophoresis (7,15,22). For a clonal infection, it is likely that E. hirae persisted in the farm, and cleaning and disinfection practices should be evaluated to minimize the risk of reoccurrence of the infection. If the bacteria are polyclonal, the source of infection would be unclear. Enterococcus hirae is commensal in the intestinal tract and is likely to be present in the gut of the chickens early in life. Translocation of E. hirae into the bloodstream should be prevented. Therefore, prevention should focus on good brooding practices, stress reduction, and preventing immunosuppressive conditions (1).

The internal biosecurity at the farms was not strict. Enterococcus hirae was diagnosed in multiple consecutive rearing periods and was present on the farm for a prolonged period; therefore, it is likely to assume that birds in all houses are exposed to E. hirae. Sex of the bird may influence susceptibility to infectious diseases; however, endocarditis was diagnosed in both male (mistakes during sexing at the hatchery) and female birds from the female line. Therefore, the influence of sex to susceptibility to E. hirae infection is less likely. On the other hand, male and female lines are of different genetic pedigrees. It may be questioned if the difference in susceptibility to infection with E. hirae between birds of the male and female lines may be explained by genetic variation.

NAbs might be a suitable trait for selective breeding for general disease resistance. NAb are heritable and beneficial effects of higher NAb titers on reduced mortality, and increased disease resistance is reported in several species (9). Laying hens with high KLH-binding NAb levels showed less lesions and mortality after inoculation with an avian pathogenic Escherichia coli. This study supported the hypothesis that KLH-binding NAb might be used as an indicator for selective breeding (11). In the present case, the opposite was observed. The affected hens had significantly higher KLH-binding NAb IgG titers compared with the unaffected males (Table 2), but more E. hirae–associated endocarditis was found in these birds. Note that differences in levels of IgM and IgG isotypes might reflect not only differences in kinetics of these antibodies but also may reflect differences in immune status (i.e., IgM antibodies have been regarded as “true” basic NAb levels), whereas IgG NAb levels may also reflect nonantigen-specific responsiveness of the infected individual. This implicates that differentiation of antibody isotypes, including time effects, is important to denote the possibility of breeding for Nab levels to reduce disease susceptibility. To summarize, this case report gives an overview of an outbreak of E. hirae–associated endocarditis in broiler breeders of the female line.

We thank A. Boon, E. de Jong, and M. Proper for technical assistance.

AV =

atrioventricular;

dph =

days posthatch;

Ig =

immunoglobulin;

KLH =

keyhole limpet hemocyanin;

NAb =

natural antibodies;

ph =

posthatch