INTRODUCTION

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Debilitating cardiopulmonary conditions in chickens have been a rising concern over the past 30 years (33). Cardiopulmonary conditions have become a serious problem in fast-growing chickens due to the complete filling of the abdominal cavity with serous fluid that leaks from pulmonary circulation. This condition is pandemic and is often referred to as "ascites" or "water belly." Research findings indicate that the cause of ascites in chickens is pulmonary hypertension syndrome (PHS) (16, 33).

This condition was first recognized in fast-growing chickens reared at high altitudes, i.e., above 3,500 m (16, 33). Birds that develop ascites do not recover; thus, the consequences are premature death or condemnation at processing (17).

Pathological and physiological evidence suggests that hypoxia activates a series of events which eventually cause PHS (9, 13, 14, 18, 37, 38, 39). The morphological symptom of PHS is right ventricular failure, often accompanied with thinning of the right ventricular wall (RVW) (2, 13, 14, 18, 27, 47). Potential explanations of hypoxia and PHS are mismatches of ventilation and perfusion in the lungs (7, 35, 48), reductions of vascular capacity in the lungs (7, 20, 21, 29, 30), hypertrophy of the RVW, an increased blood pressure via the pulmonary artery (3, 28, 36), persistent heart congestion, hydropericardium, and ascites (22).

Another physiological factor that has been associated with PHS is the voracious appetite of fast-growing broilers (4, 7). Broilers are genetically selected for hyperphagia, and this in turn causes distension of the gastrointestinal tract and decreased ventilation and perfusion of the lungs (7). Furthermore, the incidence of ascites is usually higher in broiler males than in broiler females, as well as in leghorn males (22, 24, 41). Also, the incidence of ascites can be correlated to the strain or line of broiler, maternal health, and hatchery management (1, 11, 23, 25, 26, 34, 35, 45). Ascites can also be caused by inadequate gas exchange and vasoconstriction of pulmonary arterioles (7, 8, 43). In addition, an increase in blood viscosity, caused by high altitude, rickets, respiratory disease, and reduced oxygen transfer, contributes to PHS (5, 6). Aspergillosis, caused by Aspergillus fumigatus, is thought to intensify PHS, because of its debilitating effects on respiratory tissues (15, 19).

The list of putative causes, as well as exacerbating factors, associated with PHS continues to increase. It is interesting, however, that microbial pathogens have not been implicated as a cause of PHS in any animal. Recently, Tankson et al. (J. D. Tankson, J. P. Thaxton, and Y. Vizzier-Thaxton, submitted for publication) reported that heart and lungs of broilers do not have a permanent bacterial flora; however, 41 different bacteria were found in these organs at various times before, during, and after hatching. Results suggest that these bacteria were transient and entered the heart and lungs during the hatching process, as well as during the juvenile period. Enterococcus faecalis was isolated in more chicks and at more times than any of the other 40 transient bacteria.

E. faecalis, which was previously referred to as Streptococcus faecalis, is an inhabitant of the intestinal tracts of humans and many other animals, including the chicken (12, 31). This bacterium has been described as a commensal or opportunistic, gram-positive, facultative anaerobe. When it inadvertently enters circulation, it can cause endocarditis, as well as urinary, intra-abdominal (i.a.) and pelvic infections (31). Thus, E. faecalis seemed to be a logical choice to investigate as a putative causative agent of PHS in broilers.

Pulmonary hypertension in humans is a rare, progressive and often fatal disorder (49). There are no recognized causes or cures at present. A recent estimate is that one to two people per million in the United States suffer from this condition. The National Institutes of Health (32) suggests that animal models of pulmonary hypertension are needed to understand this disease. The purposes of this paper are to evaluate E. faecalis as a cause of PHS in chickens and to offer a model for study of pulmonary hypertension in humans.

	MATERIALS AND METHODS

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Husbandry. For all trials with the exception of trial 4, chicks were brooded and reared on floor pens in a curtain-sided broiler grow-out house on pine shaving litter. Brooding heat was provided during the first 2 weeks by gas-fired heaters. A single incandescent bulb was hung in the center of each pen and it provided continuous lighting. In all trails, feed and water were available ad libitum. Birds received a starter diet containing 23% protein and 1,450 kcal of metabolizable energy (ME)/kg of body weight for the first 2 weeks. Thereafter, birds received a grower diet containing 20% protein and 1,450 kcal of ME/kg for the remainder of the experiment.

Microbiology. Enterococcus faecalis was obtained from a field strain and maintained in the laboratory by being recultured on tryptic soy agar (TSA) (Fisher Scientific Company L.L.C., Houston, Tex.) slants every 7 days. The field strain of E. faecalis was confirmed by testing it against an American Type Culture Collection (ATCC) strain of E. faecalis. This process was done by growing one ATCC dehydrated colony of E. faecalis in tryptic soy broth (TSB) (Fisher Scientific Company L.L.C.) for 24 h in an incubator at 35 to 37°C. After a viable ATCC strain of E. faecalis was established, the field strain and ATCC strain of E. faecalis were streaked onto TSA plates and placed in an incubator at 35 to 37°C for 24 h to enhance bacterial growth. Following 24 h of bacterial growth, a Gram stain was performed and the BBL Crystal Identification System (Becton Dickinson Microbiology Systems, Becton Dickinson and Co., Cockeysville, Md.) was used to identify each bacterial organism. This procedure confirmed that both the field and ATCC strains of E. faecalis were the same. The BBL Crystal Identification System was used because it provided consistent qualitative results (10, 40, 46).

Inoculation preparation. For each trial, serial dilutions (44) of a 7-day-old culture of E. faecalis were made to obtain the required dosages. Prior to preparation of the inoculum, the 7-day-old culture was grown on TSA plates for 24 h in an incubator at 35 to 37°C and reanalyzed to ensure that the culture was E. faecalis. The dilution factors used were 10⁴, 10⁵, and 10⁶. Sterile TSB was used as a diluent and as the control. In order to have enough of the bacterium to administer for each treatment, each dosage of bacteria was serially diluted into four individual tubes of TSB. The different dosages of the bacteria were placed in sterile vials whereby sterility could be maintained when the birds were challenged. Final preparation of the bacteria occurred within an hour of challenge. The amount of bacteria in 1 ml was determined by placing 1 ml from each dilution tube onto a separate TSA plate and spreading the bacteria around the plate in a figure eight motion; plates were then inverted and placed in an incubator at 35 to 37°C for 24 h. After 24 h, bacterial colonies on the plates were counted and the number obtained was multiplied by the dilution factor. The numbers of bacteria in 1 ml of each of these dilutions were 6 × 10⁶, 3 × 10⁷, and 4 × 10⁷ organisms. Since the broilers received only 0.5 ml of the dosage, the actual number of bacteria injected was half the number of bacteria found in 1 ml. So the dosages of E. faecalis were 0, 3 × 10⁶, 1.5 × 10⁷, and 2 × 10⁷.

Trial 1. In trial 1, 120 Ross × Ross chicks were obtained from an Alabama hatchery. The chicks were reared to 10 weeks of age before the study began. There were 30 birds, i.e., 15 males and 15 females, in each of four pens. In each pen, 24 birds (12 of each sex) were designated as the experimental birds, and the other 6 birds were designated as extras.

Trial 2. Trial 2 was an exact replication of trial 1 with the following exception: during necropsy, each heart was given a subjective score based upon its morphological characteristics. A score of 1 was given if the heart appeared normal and possessed normal muscular tone. A score of 2 was assigned if a cavity or depression was visible on the exterior surface of the RVW and if the heart possessed normal muscular tone. A score of 3 was assigned if the RVW exhibited the cavity and the heart was totally flaccid. Finally, a score of 4 was assigned if the cavity was present and the heart was flaccid, and upon cross-sectioning of the ventricular mass (2 to 3 mm below the lateral atrio-ventricular septum), the RVW was found to be thinned in that part of the wall where the cavity occurred. Photographs depicting hearts that received these scores are presented in Fig. 1.

View larger version (92K): [in this window] [in a new window]   FIG. 1.   Photographs of hearts to illustrate the scoring system described in this paper. (Upper left) Heart with normal appearance; score of 1. (Upper right) Cavity (*) in external surface of RVW; score of 2. (Lower left) Flaccidity (*) of RVW; score of 3. (Lower right) Vertical view of heart interior showing flaccidity plus thinning (*) of RVW; score of 4.

Trial 3. In trial 3, 150 male chicks were obtained from a commercial hatchery. Before the study began, the chicks were reared to 3 weeks of age. For this trial, the starter diet was fed continuously. A total of six pens was used, and 25 birds were allocated to each pen. Each pen contained 20 birds designated as experimental birds, and the five remaining birds were designated as extras. At 3 weeks of age, birds in three pens received 0.5 ml of TSB containing 1.5 × 10⁷ E. faecalis organisms i.a. and birds in three pens received 0.5 ml of sterile TSB i.a., which represented the control. The dosage of E. faecalis was determined in the same manner as that for trial 1. Forty-eight hours after injections, all birds were killed by cervical dislocation and necropsies were performed. During necropsy, each heart was given a score between 1 and 4, as described previously for trial 2.

Trial 4. Ninety-six Ross × Ross male chicks were obtained from a commercial hatchery and grown in heated brooder batteries for the first 3 weeks. A brooding temperature of ~35°C was provided during the first 2 weeks, while a brooding temperature of 30°C was maintained during the 3rd week. At the start of the 4th week, chicks were transferred to nonheated, metal cages for the remainder of the experimental period. Control birds were maintained in one isolation room, and E. faecalis-treated birds were maintained in another isolation room. Six birds were housed in eight cages of a battery in each isolation room. Five of the six birds in each cage served as experimental birds, whereas the other bird in each cage was an extra.

Statistical procedure. In trial 2, statistical analysis involved a two-way general analysis of variance (ANOVA). The main effects were challenge (control and E. faecalis) and dose (0, 3 × 10⁶, 1.5 × 10⁷, or 2 × 10⁷ organisms). In trials 3 and 4, a two-way ANOVA involving challenge and replication was initially used. In no case was a significant replication or replication × challenge effect found; therefore, data were pooled over replications and reanalyzed using a one-way ANOVA. Means were compared by least significant difference. The general linear model procedure of the STATISTIX analytical software (42) was employed. Statements of significance are based on a P of 0.05.

	RESULTS

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Trial 1 was conducted to assess visible changes in the hearts of fast-growing chickens that were challenged with E. faecalis. All three challenge doses, i.e., 3 × 10⁶, 1.5 × 10⁷, and 2 × 10⁷ bacteria/bird, as well as the two routes of administration, i.e., i.a. and i.v., caused a visible cavity in the external RVW (Fig. 1 [upper right panel]). As shown in Fig. 2, the average incidences of cavity occurrence were 5, 86, 100, and 95%, respectively, in the groups receiving 0, 3 × 10⁶, 1.5 × 10⁷, and 2 × 10⁷ bacteria/bird. Additionally, the average incidences of cavity formation for the two routes of administration were 93%. In controls, cavity incidence was 10% in i.a.-challenged birds and 0% in i.v.-challenged birds.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Incidence of cavity in the RVW in birds of trial 1.

In trial 2, the incidences of cavity occurrence, although not presented, were very similar to those in the groups of trial 1. Figure 3 depicts mean heart scores as quantitated by the subjective scoring system. The mean heart score in E. faecalis-challenged birds was 3.1, while this value in controls was 0.20. Mean heart scores in the i.a. and i.v. challenge groups, respectively, were 3.0 and 3.2. 

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Heart score in trial 2. Standard errors of the means ranged from 0.20 to 0.23.

Figure 4 shows mean heart scores of birds in trials 3 and 4. Mean heart scores were higher in challenged birds than in nonchallenged controls in both trials. However, differences attributable to trial 4 were not found. Specifically, the housing environment, whether on litter in a conventional poultry grow-out house or in metal cages with raised wire floors in separate rooms in an animal isolation facility, had no influence on mean heart score in both controls and challenged birds.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Heart scores in trials 3 and 4. Standard errors of the means ranged from 0.05 to 0.36. For bars labeled a and b, means within each parameter with no common superscript differ significantly (P  0.05).

	DISCUSSION

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PHS results when blood pressure in the pulmonary tree increases excessively (16, 17, 33). Back pressure causes the right ventricle of the heart to become overworked (18). This leads to right ventricular hypertrophy and thinning of the RVW (9, 17). The visual manifestation of this damage to the RVW in chickens is a cavity on the external surface of the right side of the heart (2, 13, 14, 18, 27, 47). A ratio of right ventricular weight to total ventricular weight of 0.30 or more has been proposed as the best indicator of the ascites condition in chickens (6). However, the cavity in the RVW develops before accumulation of ascites fluid in the abdominal cavity. Thus, we developed our subjective scoring system to differentiate birds challenged with E. faecalis from nonchallenged controls.

Combined results of trials 3 and 4 indicate that the mean heart score in control birds was 0.69, and this figure in challenged birds was 3.15. If a heart score of 2.0 or more is accepted as indicative of PHS caused by E. faecalis challenge, then the accuracy of this scoring method is approximately 95%.

In trials 1 to 3, both challenged and control birds were reared in a common poultry grow-out facility. It is possible that a small percentage of the controls could have experienced cross-contamination. However in trial 4, control and challenged birds were maintained in separate isolation rooms after challenge. The incidence of controls exhibiting a mean heart score of 1.5 or more was 12%.

These results suggest that cross-contamination is not an acceptable explanation for the low incidence of cavities in hearts from control birds. Other possible explanations are that bacteria other than E. faecalis affected the hearts of controls or that factors other than bacterial challenge caused the condition.

Pulmonary hypertension is a rare, progressive, and often fatal lung disorder in humans (32, 49). There are no recognized causes or cures at present. The current literature indicates that bacterial insult has not been implicated as a cause of pulmonary hypertension. Results in this report suggest that the challenge of fast-growing chickens with E. faecalis causes the major signs of PHS and may, therefore, constitute an excellent model to study the etiology of pulmonary hypertension in humans.

The accuracy of this method of identifying birds experiencing the early symptoms of PHS is satisfactory. However, this method does not attempt to delineate the various manifestations of this disease. Future studies aimed at understanding morphological and physiological characteristics associated with PHS are certainly warranted. As this model is expanded, general understanding of the etiology, as well as possible therapeutic measures and a cure for pulmonary hypertension in humans, will be augmented.