INFECTION AND IMMUNITY, Oct. 2001, p. 6318–6322 0019-9567/01/$04.00⫹0 DOI: 10.1128/IAI.69.10.6318–6322.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 69, No. 10
Pulmonary Hypertension Syndrome in Broilers Caused by Enterococcus faecalis† J. D. TANKSON,* J. P. THAXTON,
AND
Y. VIZZIER-THAXTON
Department of Poultry Science, Mississippi State University, Mississippi State, Mississippi 39762 Received 2 April 2001/Returned for modification 10 May 2001/Accepted 20 June 2001
A field strain of Enterococcus faecalis was administered to broiler chicks at doses of 0, 3 ⴛ 106, 1.5 ⴛ 107, and 2 ⴛ 107 bacteria/bird either intra-abdominally or intravenously. In trials 1 to 3, birds were reared communally in a broiler house on pine shaving litter. In trial 4, challenged and control birds were maintained in separate isolation rooms in metal cages with raised wire floors. Challenged birds exhibited a characteristic cavity or depression in the external wall of the right ventricle. A subjective scoring system was devised to quantify challenge effects by assigning each heart a score of 1 to 4. The average number of birds, over all trials and over all dose levels, exhibiting the ventricular cavity was 93%. This value in controls was 5%. The average heart score for challenged birds was 3.1, and that for controls was 0.20. Heart scores of challenged and control chicks were not different in birds reared communally or in separate isolation rooms. Additionally, both routes of administration were equally effective. Results suggest that challenge with E. faecalis caused pulmonary hypertension. 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
Debilitating cardiopulmonary conditions in chickens have been a rising concern over the past 30 years (33). Cardiopulmonary conditions have become a serious problem in fastgrowing 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
* Corresponding author. Mailing address: Poultry Science Department, Box 9665, Mississippi State, MS 39762. Phone: (662) 325-3377. Fax: (662) 325-8292. E-mail:
[email protected]. † Journal series no. J-9844 of the Mississippi Agriculture and Forestry Experiment Station (MAFES). 6318
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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 (췦) in external surface of RVW; score of 2. (Lower left) Flaccidity (췦) of RVW; score of 3. (Lower right) Vertical view of heart interior showing flaccidity plus thinning (췦) of RVW; score of 4.
a cause of PHS in chickens and to offer a model for study of pulmonary hypertension in humans. MATERIALS AND METHODS 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.
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FIG. 2. Incidence of cavity in the RVW in birds of trial 1.
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. At 10 weeks of age, 12 birds in each pen were injected i.a. with 0.5 ml of sterile TSB containing either 0, 3 ⫻ 106, 1.5 ⫻ 107, or 2 ⫻ 107 E. faecalis organisms. The other 12 experimental birds in each pen received the same dosage of E. faecalis intravenously (i.v.). Birds were spray painted on their backs to indicate challenge dosage received and route of administrations. Forty-eight hours after injections, the broilers were killed by cervical dislocation. During necropsy, hearts were removed and examined for visible morphological changes. Many chicks exhibited a depression on the surface of the RVW. Therefore, the hearts from all chicks in trial 1 were scored as positive if a depression was visible and negative if no depression was observed. The person scoring the hearts was not aware of the dosage of E. faecalis or route of administration used to treat birds. 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. 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. Both rooms were thermostatically regulated to maintain an ambient temperature of ⬃27°C. Two overhead fluorescent fixtures provided continuous light in each room.
At 5 weeks of age, five birds in each of the eight cages in the control room received 0.5 ml of sterile avian saline (0.85%) (Fisher Scientific Company L.L.C.) i.a., while five birds in each of the eight cages in the challenge room received 0.5 ml of sterile TSB containing 1.5 ⫻ 107 E. faecalis i.a. Forty-eight hours after injections, all birds in both rooms were killed, and hearts were removed within 5 min after death occurred. Birds were collected randomly and hearts were scored on a scale of 1 to 4 which was based on the previously described scoring scheme. 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 ⱕ0.05.
RESULTS 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. 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. faecalischallenged 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. 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-
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FIG. 3. Heart score in trial 2. Standard errors of the means ranged from 0.20 to 0.23.
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. DISCUSSION 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
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 ⱕ 0.05).
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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. REFERENCES 1. Arce-Menocal, J., C. Vasquez-Pelaez, C. Lopez-Coello, and E. Avila-Gonzalez. 1989. Susceptibilidad de lineas comerciales del pollo de engorde al sindrome ascitico, p. 36–41. Memorias XI Congreso Latinoamericano de Avicultura. Veterinaria Mexico, Mexico City, Mexico. 2. Cowen, B. C., R. J. Wideman, Jr., and H. Rothenbacher. 1986. Avian edema/ ascites syndrome, p. 1–30. In Proceedings of the Italian Society of Avian Pathology Conference. FECAVA, Paris, France. 3. Dale, N., and A. Villacres. 1988. Relationship of two-week body weight to the incidence of ascites in broilers. Avian Dis. 32:556–560. 4. Dunnington, E. A., and P. B. Siegel. 1996. Long-term divergent selection for eight-week body weight in White Plymouth Rock chickens. Poultry Sci. 75:1168–1179. 5. Eckert, R., D. Randall, and G. Augustine. 1988. Animal physiology. 4th ed., p. 535–548. W. H. Freeman and Co., New York, N.Y. 6. Fedde, M. R., and R. F. Wideman, Jr. 1996. Blood viscosity in broilers: influence on pulmonary hypertension syndrome. Poultry Sci. 75:1261–1267. 7. Fedde, M. R., G. E. Weigle, R. F. Wideman, Jr. 1998. Influence of feed deprivation on ventilation and gas exchange in broilers: relationship to pulmonary hypertension syndrome. Poult. Sci. 77:1704–1710. 8. Grover, R. F., J. T. Reeves, D. H. Hill, and S. G. Blount, Jr. 1963. Pulmonary vasoconstriction in steers at high altitude. J. Appl. Physiol. 18:567–574. 9. Hoerr, F. J. 1988. Pathogenesis of ascites. Poult. Dig. 47:8–12. 10. Holmes, B., M. Costas, T. Thaker, and M. Stevens. 1994. Evaluation of two BBL crystal systems for identification of some clinically important gramnegative bacteria. J. Clin. Microbiol. 32:2221–2224. 11. Huchzermeyer, F. W., A. M. C. DeRuyck, and H. Van Ark. 1988. Broiler pulmonary hypertension syndrome. III. Commercial broiler strains differ in their susceptibility. Onderstepoort J. Vet. Res. 55:5–9. 12. Jacobs, L. A., G. R. McDaniel, and C. W. Broughton. 1978. Microbial flora observed within sections of the oviduct in naturally mated, artificially inseminated, and virgin hens. Poult. Sci. 57:1550–1553. 13. Julian, R. J. 1988. Pulmonary hypertension as a cause of right ventricular failure and ascites in broilers. Zootechnia Int. 11:58–62. 14. Julian, R. J. 1990. Avian skeletal disease symposium, p. 1–11. In Annual Meeting of the American Association of Avian Pathologists. AVMA, San Antonio, Tex. 15. Julian, R. J. 1990. Cardiovascular disease, p. 345–353. In F. T. W. Jordan (ed.), Poultry diseases, 3rd ed. Bailliere Tindall, London, United Kingdom. 16. Julian, R. J. 1993. Ascites in poultry. Avian Pathol. 22:419–454. 17. Julian, R. J. 1998. Rapid growth problems: ascites and skeletal deformities in broilers. Poult. Sci. 77:1773–1780. 18. Julian, R. J., and J. B. Wilson. 1986. Right ventricular failure as a cause of ascites in broiler and roaster chickens, p. 608–611. In G. H. A. Borst (ed.), Proceedings of the 4th International Symposium of Veterinary Lab Diagnosticians, Amsterdam. Iowa State University Press, Ames, Iowa. 19. Julian, R. J., and M. Boulianne. 1988. Natural and experimental lung pathology causing pulmonary hypertension, right ventricular hypertrophy, right ventricular failure and ascites in broiler chickens. Am. J. Vet. Res. 192:209, 1781. 20. Julian, R. J., and M. Goryo. 1990. Pulmonary aspergillosis causing right ventricular failure and ascites in meat-type chickens. Avian Pathol. 19:643– 654. 21. Julian, R. J., and E. J. Squires. 1994. Haematopoietic and right ventricular response to intermittent hypobaric hypoxia in meat-type chickens. Avian Pathol. 23:539–545. 22. Julian, R. J., G. W. Friars, H. French, and M. Quinton. 1986. The relationship of right ventricular hypertrophy, right ventricular failure, and ascites to weight gain in broiler and roaster chickens. Avian Dis. 31:130–135. 23. Julian, R. J., I. McMillan, and M. Quinton. 1989. The effect of cold and dietary energy on right ventricular hypertrophy, right ventricular failure and ascites in meat-type chickens. Avian Pathol. 18:675–684. 24. Lopez-Coello, C., L. Pasch, R. Rosiles, and C. Casas. 1982. Ascites in broilers due to undetermined causes, p. 13–15. In Proceedings of the 31st Western
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