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Lesions of the pericardium and their significance in the aetiology of heart failure in broiler chickens
Research in Veterinary Science 74 (2003) 203–211 www.elsevier.com/locate/rvsc
Lesions of the pericardium and their significance in the aetiology of heart failure in broiler chickens A.A. Olkowski
a,*
, C. Wojnarowicz b, B.M. Rathgeber c, J.A. Abbott d, H.L. Classen
a
a
b
Department of Animal and Poultry Science, University of Saskatchewan, 6D34 Agriculture Building, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8 Department of Veterinary Pathology, University of Saskatchewan, 6D34 Agriculture Building, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8 c Department of Plant and Animal Sciences, Nova Scotia Agricultural College, Truro, NS, Canada B2N 5E3 d Department of Small Animal Clinical Sciences, VMRCVM, Virginia Tech, Phase II Duckpond Dr. Blacksburg, VA 24061, USA Accepted 14 December 2002
Abstract The present study focuses on lesions of the pericardium commonly observed in fast growing broilers. These lesions are examined in the context of electrophysiological and functional changes associated with cardiac performance and patho-physiology in broilers succumbing to acute or chronic heart failure. Typical lesions involving the pericardium in fast growing broiler chickens included: (1) excessive pericardial effusion, (2) locally extensive or focal adhesions between parietal and visceral components of the pericardium, (3) fibrous deposits on visceral pericardium, and (4) thickened pericardium. Echocardiographic evidence indicated that severe pericardial effusion and/or adhesions may have a restrictive effect on heart pump function, where both diastolic and systolic function of the heart may be affected. Electrocardiographic data showed a strong trend indicating that pericardial adhesions may be associated with ventricular arrhythmia and increased risk of sudden death in fast growing broilers. Relatively high levels of matrix metalloproteinase MMP-2 activity have been found in pericardial effusions from affected chickens, suggesting a possible involvement of this enzyme in the aetiology of pericardial lesions. The present results indicate that pericardial lesions may be associated with biochemical, morphological, electrophysiological, and functional changes occurring in the hearts of broilers succumbing to acute or chronic heart failure and ascites. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Pericardium; Lesions; Heart failure; Ascites; Broiler
1. Introduction Ascites syndrome and sudden death syndrome (SDS) are the most common heart related conditions in modern broiler flocks (Olkowski and Classen, 1998; Korte et al., 1999). Morbidities, mortalities, and carcass condemnations associated with heart failure are the causes of significant economic losses in the broiler industry throughout the world (Maxwell and Robertson, 1997, 1998; Jakob et al., 1998). The natural history of events associated with acute or chronic heart failure in broilers is not clear. The existing evidence suggests that pathological changes in the ven*
Corresponding author. Tel.: +1-306-966-5848; fax: +1-306-9666598. E-mail address: [email protected] (A.A. Olkowski).
tricular myocardium underlie the functional impairment leading to acute heart failure and sudden death, or in chronic cases, myocardial dysfunction leads to impaired systolic performance, ventricular dilatation, and ultimately signs of congestive heart failure. Focal myocardial necrosis, scarring, and arteriosclerotic changes have been found in the hearts of chickens dying of sudden death syndrome (Kawada et al., 1994). Electron microscope studies have shown that the architecture of cardiac extra-cellular matrix (ECM) and sarcomeres is severely disturbed in broilers with fulminant heart failure and ascites (Olkowski et al., 2001). However, the primary causes of the changes in the myocardium in birds succumbing to acute or chronic heart failure remains obscure. Necropsy findings such as hydropericardium (i.e., pericardial effusion) and epicardial fibrosis have been
0034-5288/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0034-5288(03)00004-3
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described in broilers dying from heart failure (Nakamura et al., 1999). Studies conducted in the authors laboratory over the last few years (Olkowski et al., unpublished observations) showed similar findings, but we also observed that significant pericardial and myocardial lesions indicative of sub-clinical heart disease are commonly seen in many fast growing broilers without overt clinical signs. Pericardial lesions may be of patho-physiological significance in several aspects of heart failure pathogenesis. For instance, excessive accumulation of serous fluid in pericardial space (hyropericardium) may lead to impairment of diastolic function. Further, pericardial lesions may create a milieu conducive to electrophysiological disturbances in the heart and arrhythmia (Ware et al., 2000). In this context, it is noteworthy that fast growing broilers are highly prone to cardiac dysrhythmia (Grashorn, 1994; Olkowski et al., 1997; Olkowski and Classen, 1998; Korte et al., 1999), and the clinical course of both SDS and ascites appear to be closely associated with cardiac arrhythmias (Grashorn, 1994; Olkowski and Classen, 1998; Korte et al., 1999). The present work examines gross pathological and histopathological aspects of pericardial lesions in the context of biochemical, electrophysiological, and functional characteristics observed in broilers succumbing to acute or chronic heart failure.
Broilers showing clinical signs of heart failure, with or without ascites, were examined at the onset of clinical signs. Echocardiographic examination was performed with subjects lightly anaesthetized with isofluorane using a diagnostic echocardiograph (Hewlett-Packard 5500). A 3.5 MHz transducer was utilized for two-dimensional echocardiography and for Doppler colour flow imaging. All birds were monitored daily for overt signs of heart failure and ascites. The birds with advanced clinical signs in extremis were euthanazed. Birds that died or were euthanazed during the course of the study, and all surviving birds at the completion of clinical studies were subjected to routine post-mortem evaluation. 2.3. Light microscopy The hearts from two broilers that died suddenly showing severe focal pericardial adhesions upon gross necropsy were subjected to detailed histopathological examination. The hearts were removed and processed immediately after death. Following the fixation in formaldehyde buffer, blocks of myocardium with attached pericardial adhesions were embedded in paraffin wax. Sections (5 lm) taken midway through the lesion were processed for light microscopy and stained with haematoxylin and eosin, and MassonÕs stain. 2.4. Pericardial fluid bacteriology
2. Materials and methods 2.1. General Four separate flocks, each comprising 200–300 commercial male broilers, were studied in order to correlate observations from clinical evaluation, echocardiographic examination, ECG data, and post-mortem examination of heart lesions. Experimental details, management of animals, and feeding regimes were as described previously (Olkowski et al., 1999). 2.2. Clinical studies Randomly selected birds from each flock were used in clinical studies. The clinical data were collected between the 4th and 6th week of life. The ECG recordings were performed as previously described (Olkowski et al., 1997). A total of 30 broilers with fulminant heart failure and ascites, and 50 normal broilers were examined using echocardiography. In order to evaluate whether pericardial lesions are associated with the natural history of heart failure and ascites, we also examined 15 apparently normal fast growing broilers during the 4th week of life, and 5 broilers showing clinical signs of heart failure but no ascites. These signs included: fatigue, exercise intolerance, increased respiration, and cyanosis.
Pericardial fluid from 20 broilers showing significant pericardial effusion upon post-mortem examination was collected into sterile syringes. The samples were inoculated into a Brain Heart Infusion (BHI) liquid medium (Becton–Dickinson, MD, USA), and incubated at 37 °C for 48 h. The BHI medium is used in routine evaluation of clinical material for wide variety of microorganisms including bacteria, yeasts, and molds. 2.5. Zymography Samples of pericardial fluid and blood plasma from leghorns (resistant to ascites), slow growing feed restricted broilers (low risk of ascites), fast growing ad libitum fed broilers (high risk of ascites), and ascitic broilers were screened for matrix metalloproteinases (MMP) activity. Leghorn chickens were used as a physiological reference. The activity of MMPs in blood plasma and pericardial fluid was measured as previously described (Olkowski et al., 2001). Briefly, samples were diluted 1:4 and loaded on 8% SDS–PAGE gels in which the separating gels were co-polymerized with 2 mg/ml gelatin (Sigma, St. Louis, MO). After electrophoresis, the gels were washed with 2.5% Triton X-100, and then incubated in incubation buffer (0.15 M NaCl, 5 mM CaCl2 , 0.05% NaN3 , and 50 mM Tris–HCl buffer, pH 7.5) for 16 h. After incubation, the gels were stained with
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0.05% Coomassie brilliant blue G-250 (Sigma) in a mixture of methanol:acetic acid:water (2.5:1:6.5) and destained in 4% methanol with 8% acetic acid. The gelatinolytic activities were detected as transparent bands against the background of Coomassie brilliant blue-stained gelatin. To evaluate the activities of the detected enzymes, the intensities of the separate bands on zymograms were analysed using an IS-1000 digital imaging system (Alpha Innotech, San Leonardo, CA). The experimental protocols were approved by the University of Saskatchewan Animal Care Committee
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and the procedures were performed in accordance with the requirements of the Guide to the Care and Use of Experimental Animals (Canadian Council On Animal Care, 1993). 2.6. Statistical analysis Statistical analyses were carried out by analysis of variance from the microcomputer package Number Cruncher Statistical System (Hintze, 1995). Means were compared using FisherÕs least significant difference test.
Table 1 Comparison of lesion involving pericardium in broilers with fulminant heart failure and ascites and in broilers without overt clinical signs of heart failure in which lesions indicative of sub-clinical heart disease were detected at necropsy Flock
1. (n ¼ 300) 2. (n ¼ 200) 3. (n ¼ 250) 4. (n ¼ 200)
Broilers with fulminat heart failure and ascites
Broilers with sub-clinical heart condition
Birds affected
Birds affected
Pericardial effusion
Pericardial adhesions
88 (29.3) 44 (22.0) 76 (30.4) 58 (29.0)
58 (65.9) 41 (93.2) 74 (97.4) 42 (72.4)
14 [2] (15.9) 8 [0] (18.2) 22 [2] (28.9) 9 [1] (15.5)
a
78 (26.0)b 49 (24.5) 55 (22.0) 41 (20.5)
Pericardial effusion 73 (93.6)c 48 (98.0) 55 (100) 40 (97.6)
Pericardial adhesions d
31 [5] (39.7)c 12 [2] (24.5) 26 [5] (47.3) 12 [2] (29.3)
n, number of birds. Frequency of occurrence. b Percentage of population. c Percent affected. d Total affected, numbers in square parenthesis indicate cases with severe focal adhesions. a
Fig. 1. Lesions associated with the pericardium in broiler chicken. Typical changes in the heart from ascitic chicken (a) include a prominent pericardial effusion (large arrow), and locally extensive lesions where parietal pericardium adheres to the visceral pericardium (small arrows). Locally extensive scars remnant after separation of pericardial adhesion in ascitic broiler (b) and a broiler with moderate left ventricular dilation (c).
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Statistical significance was assumed to exist when the probability of making a type I error was less than 0.05.
3. Results Prominent pathological changes involving the pericardium and myocardium were observed in the hearts from all birds that developed ascites, but similar lesions indicative of sub-clinical heart disease were also seen in 20–30% of broiler population without overt clinical signs of heart failure (Table 1). Characteristic gross pathological changes involving the myocardium included left and right ventricular, and atrial dilatation, and atrio-ventricular valve degeneration. Typical lesions involving the pericardium included: (1) excessive accu-
mulation of serous fluid in the pericardial space, (2) locally extensive or focal adhesions between parietal and visceral components of the pericardium, (3) focal or locally extensive fibrous deposits on visceral pericardium that may or may not have been associated with adhesions, and (4) thickened pericardium. In a typical case of chronic heart failure and ascites, as well as in many fast growing broilers without ascites, a very characteristic finding was a prominent pericardial effusion (PE) (Fig. 1). In broilers with advanced pathological changes in the heart with or without ascites, the volume of PE ranged from 5 to 15 ml, and in broilers showing sub-clinical heart lesions, PE was between 3 and 10 ml. In contrast, the amount of fluid in the pericardial space of normal chickens was between 0.5 and 1.5 ml. Based on post-mortem examination more than
Fig. 2. Severe, focal pericardial adhesion in broiler chicken. Note parietal pericardium firmly attached in several spots (a), and a deep erosion (arrow) remaining upon forceful separation of adhesion (b).
Fig. 3. Two-dimensional echocardiagraphic images of heart from broilers with fulminant heart failure and ascites. Cardiac structures are identified as follows LV (left ventriculum), RV (right ventriculum), LA (left atrium), and RA (right atrium). Noteworthy are extensive PE and associated collapsing of left atrial wall (a), and substantial compression of free left ventricular wall (b) during diastole.
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90% of broilers with ascites showed severe PE. Moderate to severe PE were seen in 20–30% of broilers without overt clinical signs of heart failure.
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Pericardial effusion was frequently accompanied by locally extensive, and/or focal adhesions between parietal and visceral pericardium (Fig. 1a). Some 20–40% of
Fig. 4. Two-dimensional imaging from a broiler chicken with ascites. Colour flow Doppler mapping was used to detect blood flow disturbances. Cardiac structures are identified in (a) as follows: LV (left ventriculum), RV (right ventriculum), LA (left atrium), and A (aorta). Blood flow patterns are identified by colours, where red depicts ventricular inflow and blue depicts ventricular outflow. Noteworthy is a distortion of left ventricle geometry (a) associated with an adhesion between parietal and visceral pericardium (arrow). Distortion is most pronounced at the end of systole (a), beginning of diastole (b), and persist though mid diastole (c). Bi-ventricular dilatation is clearly visible during end of diastole (d). Distortion of left ventricle geometry becomes apparent during early systole (e) and progresses through end of systole (f), whereas malfunction of the left A-V valve apparatus is evidenced by blood regurgitating back to left atrium (arrow). Focal pericardial adhesions between parietal and visceral pericardium at the apex were seen at post-mortem examination.
Fig. 5. Microscopic lesions in the pericardium and myocardium from a broiler chicken that died suddenly. Ventricular arrhythmia (premature ventricular contractions) was observed in this bird 3 days prior to death. Note a significant mass of the tissue arising from the epicardium, with a core consisting of fibrous tissue, and collagen strands penetrating (arrows) the mural myocardium (a). The myocardium close to the lumen of the left ventricle (b) showed several random discrete foci of myofiber disorganization and fragmentation (arrows).
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ascitic broilers, and 10–30% of apparently normal broilers showed adhesions between the parietal and visceral pericardium or fibrous deposits on visceral pericardium. In most instances, adhesions appeared in the form of lightly attached parietal and visceral pericardium (Fig. 1a), leaving a shallow scar upon separation (Figs. 1b and c). More severe pericardial adhesions occur in approximately 2–10% of broilers. In severe cases, the pericardial remnants were firmly attached in several spots (Fig. 2a), leaving a considerable erosion upon separation, which sometimes required considerable force (Fig. 2b). All ascitic broilers examined using echocardiography showed PE, and in 90% of these birds, PE was severe. Of the 15 broilers examined during the 4th week, 4 developed ascites during the 5th or 6th week of life and all showed a history of PE. All five broilers with signs of heart failure but no ascites, showed PE upon initial echocardiographic examination, and three of them developed ascites. In four cases with severe PE collapsing
of the atrial and/or compression of left ventricular free wall was evident (Figs. 3a and b). In three broilers, the presence of severe adhesions was apparent on echocardiographic images. Colour Doppler flow mapping revealed that adhesions may affect systolic and diastolic function (Fig. 4). In all cases where pericardial lesions were identified during echocardiographic examination, the presence of these lesions was corroborated by postmortem findings. Among the 30 broilers examined with ECG, in which severe pericardial lesions were observed at necropsy, 14 showed cardiac arrhythmia. Eight broilers that died suddenly had severe, focal pericardial adhesions, and five of them had a history of ventricular arrhythmia. Histopathological changes in the pericardium and myocardium of one of these birds are presented in Fig. 5a and b. The fluid accumulated in the pericardial space was clear, light yellow, and had the appearance of non-inflammatory transudate. No bacteria were recovered
Fig. 6. Matrix collagenase activity in chicken pericardial fluid and blood plasma examined using gelatin zymography. (a) Representative zymograms of pericardial fluid and blood plasma from normal chickens, chicken with sub-clinical heart disease, and ascitic chickens. The column labelled MMP STD indicates molecular markers of MMP-9 (92 kDa), pro-MMP-2 (72 kDa), and active MMP-2 (69 kDa) obtained from cell culture medium of the human fibrosarcoma HT 1080 cell line. The activity of MMPs in the pericardial fluid and plasma revealed the presence of both pro-MMP-2 (72 kDa), and active MMP-2 (69 kDa), but MMP-9 (92 kDa) was absent. (b) Trends in relative gelatinolytic activity of MMP-2, pro-enzyme, and the activated form in pericardial fluid and blood plasma from leghorns (resistant to ascites), slow growing feed restricted broilers (Broiler R, low risk of ascites), fast growing ad libitum fed broilers (Broiler A-L, high risk of ascites), and broilers with heart failure and ascites (CHF-Ascites). The values represent means (SE) from 2 leghorns, 4 slow growing broilers, 7 fast growing broilers, and 4 ascitic broilers.
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from the pericardial fluid. The study of activity of MMPs in blood plasma and pericardial fluid revealed the presence of both pro-MMP-2 (72 kDa), and active MMP-2 (69 kDa), but MMP-9 (92 kDa) was virtually absent. Fig. 6a shows typical zymograms from normal and abnormal individuals. The relative gelatinolytic activity of MMP-2, in particular the activated form, was higher (p < 0:05) in pericardial fluid from broilers (generally prone to heart failure and ascites) in comparison to leghorns (resistant to heart disease), and was the highest (all p < 0:05) in birds with fulminat heart failure and ascites (Fig. 6b). Noteworthy are relatively higher pro-MMP-2 activities in fast growing broilers (high risk of ascites) in comparison to slow growing broilers (low risk of ascites). Generally, the active form of MMP-2 was higher in PE than in blood plasma of broiler chickens.
4. Discussion From the study of Nakamura et al. (1999), our retrospective data (Olkowski et al., unpublished observations), and the present results it is evident that lesions in the pericardium occur in the vast majority (>90%) of broilers succumbing to heart failure and ascites. Based on echocardiographic observations and post-mortem data, it appears that pericardial lesions represent early changes associated with the development of heart failure and ascites in broilers, but because pericardial lesions almost always occur together with pathological changes in the myocardium, their aetiological identity it is not clear at present. Because of the close anatomical relationship with the myocardium, the lesions associated with the pericardium may have a wide range of possible adverse effects on cardiac muscle patho-physiology and heart pump performance. Therefore, the pericardial lesions described here ought to be considered in the context of their potential effects on the mechanical performance of the heart pump, as well as from the perspective of electrophysiological, functional, and biochemical characteristics. Based on echocardiagraphic examination, it is apparent that pericardial lesions are present before overt signs of heart failure appear. Notably, pericardial adhesions may affect cardiac diastolic and systolic function, whereas excess fluid in the pericardial space may increase the haemodynamic burden of the heart mainly during diastole. The restrictive effect of PE and/or pericardial adhesions therefore ought to be considered in the aetiology of heart pump failure. Poor performance of the heart pump in broilers with fulminant heart failure and ascites is clinically evidenced by impaired ventricular wall motion, reduced fractional shortening, and reduced cardiac output (Olkowski and
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Classen, 1999; Olkowski et al., 1999). All these changes are consistent with the restrictive effects of PE. Increased haemodynamic burden in heart chambers associated with excessive pressure in the pericardial space may contribute to the rise in hydrostatic pressure in the venous circulation, and therefore may be one of the more significant factors contributing to the formation of ascites in affected broilers. In comparison to other classes of chickens, fast growing broilers show a high incidence of cardiac dysrhythmia (Grashorn, 1994; Olkowski et al., 1997; Olkowski and Classen, 1998; Korte et al., 1999). The clinical course of both acute heart failure (SDS) and chronic heart failure and ascites appears to be closely associated with cardiac arrhythmias (Grashorn, 1994; Olkowski and Classen, 1997, 1998; Korte et al., 1999). The increased susceptibility of the broiler heart to arrhythmic activity may be associated with lesions in the pericardium as well as in the myocardium. There is little doubt that lesions in the myocardium may compromise electrical stability of the heart and lead to fatal arrhythmia. However, lesions of the pericardium in broilers may also increase the risk of arrhythmic activity. The issue of pericardial lesions as a potential arrhythmogenic factor in fast growing broilers deserves attention for at least two reasons: (1) because of the central role of the epicardial surfaces of the ventricles in the propagation of the depolarization wave (Kisch, 1951; Moore, 1965), and (2) cardiac irritability is higher in fast growing broilers in comparison to slower growing broilers (Greenlees et al., 1989). Injury in the pericardium may create a milieu conducive to electrophysiological disturbances in the heart and arrhythmia (Ware et al., 2000). Also, spatial/temporal non-uniformity, and the multi-focal nature of depolarization of the epicardial surfaces of the ventricles in the chicken (Kharin et al., 2001) may further increase the periods of electrical instability of the heart. This may be of importance in arrhythmogenesis as well as in the process of degeneration of arrhythmic episodes into fatal ventricular fibrillation (Olkowski and Classen, 1997). The pathological process that initiates pericardial effusion, adhesions and/or epicardial surface fibrosis is not known, but in view of the present findings may be associated with excessive activity of MMP-2. The importance of MMPs in myocardial pathological remodeling has been documented (Dollery et al., 1995; Coker et al., 1999; Peterson et al., 2001). Our previous study (Olkowski et al., 2001) indicated that the pathogenesis of heart failure and ascites in fast growing broilers is associated with the increased activity of MMP-2 in heart tissue. However, considering that in the present study higher levels of activated MMP-2 was seen in PE of ascitic birds as well as in broilers with pathological evidence of sub-clinical heart disease, it can be inferred
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that increased MMP activity is a factor that appears early in the development of heart failure and ascites. Further, it is noteworthy that the MMP-2 levels (the active form) in broilers, was higher in PE fluid than in blood plasma. Thus, the local synthesis and/or activation of MMPs may be aetiologically associated with the development of pericardial lesions, and may play a significant role in the aetiology of heart failure and ascites in broilers. Collagen degradation in soft connective tissue is mediated mainly by MMPs, with MMP-2 playing a key role in this process (Aimes and Quigley, 1995; Creemers et al., 1998). It is becoming increasingly clear that MMPs are involved in various aspects of physiological and pathological vascular remodeling (for review see Galis and Khatri, 2002). Hypothetically, the breakdown of ECM may alter the passive properties of blood vessels, which could lead to increased vascular permeability, thus facilitating PE and ascites in predisposed broilers. The reason why fast growing broilers show higher levels of MMP activity in blood plasma is unknown. One of the more plausible explanations is that excessive levels of MMP may be directly associated with the rapid growth potential of broiler chickens. It has been shown that the activation and/or synthesis of MMPs may be regulated by growth factors, cytokines and hormones (Tyagi et al., 1995; Rooprai et al., 2000). Hence, the very factors that are essential for growth may mediate excessive synthesis of MMP-2 in fast growing broiler chickens. The increased activity of MMPs in some fast growing broilers may be viewed as a factor increasing the risk of heart failure and ascites. In summary, the present study confirms the fact that pericardial lesions are involved in the natural history of cardiac failure in broiler chickens. For this reason, the pathogenesis of pericardial lesions themselves warrants thorough investigation. Better understanding of the relationship between PE, adhesions, and myocardial structural abnormalities, and their electrophysiological and haemodynamic significance may help to unravel the mechanisms involved in the development of both acute and chronic heart failure in broilers.
Acknowledgements The research presented in this paper has been supported by grants provided by the Poultry Industry Council.
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Lesions of the pericardium and their significance in the aetiology of heart failure in broiler chickens A.A. Olkowski
a,*
, C. Wojnarowicz b, B.M. Rathgeber c, J.A. Abbott d, H.L. Classen
a
a
b
Department of Animal and Poultry Science, University of Saskatchewan, 6D34 Agriculture Building, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8 Department of Veterinary Pathology, University of Saskatchewan, 6D34 Agriculture Building, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8 c Department of Plant and Animal Sciences, Nova Scotia Agricultural College, Truro, NS, Canada B2N 5E3 d Department of Small Animal Clinical Sciences, VMRCVM, Virginia Tech, Phase II Duckpond Dr. Blacksburg, VA 24061, USA Accepted 14 December 2002
Abstract The present study focuses on lesions of the pericardium commonly observed in fast growing broilers. These lesions are examined in the context of electrophysiological and functional changes associated with cardiac performance and patho-physiology in broilers succumbing to acute or chronic heart failure. Typical lesions involving the pericardium in fast growing broiler chickens included: (1) excessive pericardial effusion, (2) locally extensive or focal adhesions between parietal and visceral components of the pericardium, (3) fibrous deposits on visceral pericardium, and (4) thickened pericardium. Echocardiographic evidence indicated that severe pericardial effusion and/or adhesions may have a restrictive effect on heart pump function, where both diastolic and systolic function of the heart may be affected. Electrocardiographic data showed a strong trend indicating that pericardial adhesions may be associated with ventricular arrhythmia and increased risk of sudden death in fast growing broilers. Relatively high levels of matrix metalloproteinase MMP-2 activity have been found in pericardial effusions from affected chickens, suggesting a possible involvement of this enzyme in the aetiology of pericardial lesions. The present results indicate that pericardial lesions may be associated with biochemical, morphological, electrophysiological, and functional changes occurring in the hearts of broilers succumbing to acute or chronic heart failure and ascites. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Pericardium; Lesions; Heart failure; Ascites; Broiler
1. Introduction Ascites syndrome and sudden death syndrome (SDS) are the most common heart related conditions in modern broiler flocks (Olkowski and Classen, 1998; Korte et al., 1999). Morbidities, mortalities, and carcass condemnations associated with heart failure are the causes of significant economic losses in the broiler industry throughout the world (Maxwell and Robertson, 1997, 1998; Jakob et al., 1998). The natural history of events associated with acute or chronic heart failure in broilers is not clear. The existing evidence suggests that pathological changes in the ven*
Corresponding author. Tel.: +1-306-966-5848; fax: +1-306-9666598. E-mail address: [email protected] (A.A. Olkowski).
tricular myocardium underlie the functional impairment leading to acute heart failure and sudden death, or in chronic cases, myocardial dysfunction leads to impaired systolic performance, ventricular dilatation, and ultimately signs of congestive heart failure. Focal myocardial necrosis, scarring, and arteriosclerotic changes have been found in the hearts of chickens dying of sudden death syndrome (Kawada et al., 1994). Electron microscope studies have shown that the architecture of cardiac extra-cellular matrix (ECM) and sarcomeres is severely disturbed in broilers with fulminant heart failure and ascites (Olkowski et al., 2001). However, the primary causes of the changes in the myocardium in birds succumbing to acute or chronic heart failure remains obscure. Necropsy findings such as hydropericardium (i.e., pericardial effusion) and epicardial fibrosis have been
0034-5288/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0034-5288(03)00004-3
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described in broilers dying from heart failure (Nakamura et al., 1999). Studies conducted in the authors laboratory over the last few years (Olkowski et al., unpublished observations) showed similar findings, but we also observed that significant pericardial and myocardial lesions indicative of sub-clinical heart disease are commonly seen in many fast growing broilers without overt clinical signs. Pericardial lesions may be of patho-physiological significance in several aspects of heart failure pathogenesis. For instance, excessive accumulation of serous fluid in pericardial space (hyropericardium) may lead to impairment of diastolic function. Further, pericardial lesions may create a milieu conducive to electrophysiological disturbances in the heart and arrhythmia (Ware et al., 2000). In this context, it is noteworthy that fast growing broilers are highly prone to cardiac dysrhythmia (Grashorn, 1994; Olkowski et al., 1997; Olkowski and Classen, 1998; Korte et al., 1999), and the clinical course of both SDS and ascites appear to be closely associated with cardiac arrhythmias (Grashorn, 1994; Olkowski and Classen, 1998; Korte et al., 1999). The present work examines gross pathological and histopathological aspects of pericardial lesions in the context of biochemical, electrophysiological, and functional characteristics observed in broilers succumbing to acute or chronic heart failure.
Broilers showing clinical signs of heart failure, with or without ascites, were examined at the onset of clinical signs. Echocardiographic examination was performed with subjects lightly anaesthetized with isofluorane using a diagnostic echocardiograph (Hewlett-Packard 5500). A 3.5 MHz transducer was utilized for two-dimensional echocardiography and for Doppler colour flow imaging. All birds were monitored daily for overt signs of heart failure and ascites. The birds with advanced clinical signs in extremis were euthanazed. Birds that died or were euthanazed during the course of the study, and all surviving birds at the completion of clinical studies were subjected to routine post-mortem evaluation. 2.3. Light microscopy The hearts from two broilers that died suddenly showing severe focal pericardial adhesions upon gross necropsy were subjected to detailed histopathological examination. The hearts were removed and processed immediately after death. Following the fixation in formaldehyde buffer, blocks of myocardium with attached pericardial adhesions were embedded in paraffin wax. Sections (5 lm) taken midway through the lesion were processed for light microscopy and stained with haematoxylin and eosin, and MassonÕs stain. 2.4. Pericardial fluid bacteriology
2. Materials and methods 2.1. General Four separate flocks, each comprising 200–300 commercial male broilers, were studied in order to correlate observations from clinical evaluation, echocardiographic examination, ECG data, and post-mortem examination of heart lesions. Experimental details, management of animals, and feeding regimes were as described previously (Olkowski et al., 1999). 2.2. Clinical studies Randomly selected birds from each flock were used in clinical studies. The clinical data were collected between the 4th and 6th week of life. The ECG recordings were performed as previously described (Olkowski et al., 1997). A total of 30 broilers with fulminant heart failure and ascites, and 50 normal broilers were examined using echocardiography. In order to evaluate whether pericardial lesions are associated with the natural history of heart failure and ascites, we also examined 15 apparently normal fast growing broilers during the 4th week of life, and 5 broilers showing clinical signs of heart failure but no ascites. These signs included: fatigue, exercise intolerance, increased respiration, and cyanosis.
Pericardial fluid from 20 broilers showing significant pericardial effusion upon post-mortem examination was collected into sterile syringes. The samples were inoculated into a Brain Heart Infusion (BHI) liquid medium (Becton–Dickinson, MD, USA), and incubated at 37 °C for 48 h. The BHI medium is used in routine evaluation of clinical material for wide variety of microorganisms including bacteria, yeasts, and molds. 2.5. Zymography Samples of pericardial fluid and blood plasma from leghorns (resistant to ascites), slow growing feed restricted broilers (low risk of ascites), fast growing ad libitum fed broilers (high risk of ascites), and ascitic broilers were screened for matrix metalloproteinases (MMP) activity. Leghorn chickens were used as a physiological reference. The activity of MMPs in blood plasma and pericardial fluid was measured as previously described (Olkowski et al., 2001). Briefly, samples were diluted 1:4 and loaded on 8% SDS–PAGE gels in which the separating gels were co-polymerized with 2 mg/ml gelatin (Sigma, St. Louis, MO). After electrophoresis, the gels were washed with 2.5% Triton X-100, and then incubated in incubation buffer (0.15 M NaCl, 5 mM CaCl2 , 0.05% NaN3 , and 50 mM Tris–HCl buffer, pH 7.5) for 16 h. After incubation, the gels were stained with
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0.05% Coomassie brilliant blue G-250 (Sigma) in a mixture of methanol:acetic acid:water (2.5:1:6.5) and destained in 4% methanol with 8% acetic acid. The gelatinolytic activities were detected as transparent bands against the background of Coomassie brilliant blue-stained gelatin. To evaluate the activities of the detected enzymes, the intensities of the separate bands on zymograms were analysed using an IS-1000 digital imaging system (Alpha Innotech, San Leonardo, CA). The experimental protocols were approved by the University of Saskatchewan Animal Care Committee
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and the procedures were performed in accordance with the requirements of the Guide to the Care and Use of Experimental Animals (Canadian Council On Animal Care, 1993). 2.6. Statistical analysis Statistical analyses were carried out by analysis of variance from the microcomputer package Number Cruncher Statistical System (Hintze, 1995). Means were compared using FisherÕs least significant difference test.
Table 1 Comparison of lesion involving pericardium in broilers with fulminant heart failure and ascites and in broilers without overt clinical signs of heart failure in which lesions indicative of sub-clinical heart disease were detected at necropsy Flock
1. (n ¼ 300) 2. (n ¼ 200) 3. (n ¼ 250) 4. (n ¼ 200)
Broilers with fulminat heart failure and ascites
Broilers with sub-clinical heart condition
Birds affected
Birds affected
Pericardial effusion
Pericardial adhesions
88 (29.3) 44 (22.0) 76 (30.4) 58 (29.0)
58 (65.9) 41 (93.2) 74 (97.4) 42 (72.4)
14 [2] (15.9) 8 [0] (18.2) 22 [2] (28.9) 9 [1] (15.5)
a
78 (26.0)b 49 (24.5) 55 (22.0) 41 (20.5)
Pericardial effusion 73 (93.6)c 48 (98.0) 55 (100) 40 (97.6)
Pericardial adhesions d
31 [5] (39.7)c 12 [2] (24.5) 26 [5] (47.3) 12 [2] (29.3)
n, number of birds. Frequency of occurrence. b Percentage of population. c Percent affected. d Total affected, numbers in square parenthesis indicate cases with severe focal adhesions. a
Fig. 1. Lesions associated with the pericardium in broiler chicken. Typical changes in the heart from ascitic chicken (a) include a prominent pericardial effusion (large arrow), and locally extensive lesions where parietal pericardium adheres to the visceral pericardium (small arrows). Locally extensive scars remnant after separation of pericardial adhesion in ascitic broiler (b) and a broiler with moderate left ventricular dilation (c).
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Statistical significance was assumed to exist when the probability of making a type I error was less than 0.05.
3. Results Prominent pathological changes involving the pericardium and myocardium were observed in the hearts from all birds that developed ascites, but similar lesions indicative of sub-clinical heart disease were also seen in 20–30% of broiler population without overt clinical signs of heart failure (Table 1). Characteristic gross pathological changes involving the myocardium included left and right ventricular, and atrial dilatation, and atrio-ventricular valve degeneration. Typical lesions involving the pericardium included: (1) excessive accu-
mulation of serous fluid in the pericardial space, (2) locally extensive or focal adhesions between parietal and visceral components of the pericardium, (3) focal or locally extensive fibrous deposits on visceral pericardium that may or may not have been associated with adhesions, and (4) thickened pericardium. In a typical case of chronic heart failure and ascites, as well as in many fast growing broilers without ascites, a very characteristic finding was a prominent pericardial effusion (PE) (Fig. 1). In broilers with advanced pathological changes in the heart with or without ascites, the volume of PE ranged from 5 to 15 ml, and in broilers showing sub-clinical heart lesions, PE was between 3 and 10 ml. In contrast, the amount of fluid in the pericardial space of normal chickens was between 0.5 and 1.5 ml. Based on post-mortem examination more than
Fig. 2. Severe, focal pericardial adhesion in broiler chicken. Note parietal pericardium firmly attached in several spots (a), and a deep erosion (arrow) remaining upon forceful separation of adhesion (b).
Fig. 3. Two-dimensional echocardiagraphic images of heart from broilers with fulminant heart failure and ascites. Cardiac structures are identified as follows LV (left ventriculum), RV (right ventriculum), LA (left atrium), and RA (right atrium). Noteworthy are extensive PE and associated collapsing of left atrial wall (a), and substantial compression of free left ventricular wall (b) during diastole.
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90% of broilers with ascites showed severe PE. Moderate to severe PE were seen in 20–30% of broilers without overt clinical signs of heart failure.
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Pericardial effusion was frequently accompanied by locally extensive, and/or focal adhesions between parietal and visceral pericardium (Fig. 1a). Some 20–40% of
Fig. 4. Two-dimensional imaging from a broiler chicken with ascites. Colour flow Doppler mapping was used to detect blood flow disturbances. Cardiac structures are identified in (a) as follows: LV (left ventriculum), RV (right ventriculum), LA (left atrium), and A (aorta). Blood flow patterns are identified by colours, where red depicts ventricular inflow and blue depicts ventricular outflow. Noteworthy is a distortion of left ventricle geometry (a) associated with an adhesion between parietal and visceral pericardium (arrow). Distortion is most pronounced at the end of systole (a), beginning of diastole (b), and persist though mid diastole (c). Bi-ventricular dilatation is clearly visible during end of diastole (d). Distortion of left ventricle geometry becomes apparent during early systole (e) and progresses through end of systole (f), whereas malfunction of the left A-V valve apparatus is evidenced by blood regurgitating back to left atrium (arrow). Focal pericardial adhesions between parietal and visceral pericardium at the apex were seen at post-mortem examination.
Fig. 5. Microscopic lesions in the pericardium and myocardium from a broiler chicken that died suddenly. Ventricular arrhythmia (premature ventricular contractions) was observed in this bird 3 days prior to death. Note a significant mass of the tissue arising from the epicardium, with a core consisting of fibrous tissue, and collagen strands penetrating (arrows) the mural myocardium (a). The myocardium close to the lumen of the left ventricle (b) showed several random discrete foci of myofiber disorganization and fragmentation (arrows).
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ascitic broilers, and 10–30% of apparently normal broilers showed adhesions between the parietal and visceral pericardium or fibrous deposits on visceral pericardium. In most instances, adhesions appeared in the form of lightly attached parietal and visceral pericardium (Fig. 1a), leaving a shallow scar upon separation (Figs. 1b and c). More severe pericardial adhesions occur in approximately 2–10% of broilers. In severe cases, the pericardial remnants were firmly attached in several spots (Fig. 2a), leaving a considerable erosion upon separation, which sometimes required considerable force (Fig. 2b). All ascitic broilers examined using echocardiography showed PE, and in 90% of these birds, PE was severe. Of the 15 broilers examined during the 4th week, 4 developed ascites during the 5th or 6th week of life and all showed a history of PE. All five broilers with signs of heart failure but no ascites, showed PE upon initial echocardiographic examination, and three of them developed ascites. In four cases with severe PE collapsing
of the atrial and/or compression of left ventricular free wall was evident (Figs. 3a and b). In three broilers, the presence of severe adhesions was apparent on echocardiographic images. Colour Doppler flow mapping revealed that adhesions may affect systolic and diastolic function (Fig. 4). In all cases where pericardial lesions were identified during echocardiographic examination, the presence of these lesions was corroborated by postmortem findings. Among the 30 broilers examined with ECG, in which severe pericardial lesions were observed at necropsy, 14 showed cardiac arrhythmia. Eight broilers that died suddenly had severe, focal pericardial adhesions, and five of them had a history of ventricular arrhythmia. Histopathological changes in the pericardium and myocardium of one of these birds are presented in Fig. 5a and b. The fluid accumulated in the pericardial space was clear, light yellow, and had the appearance of non-inflammatory transudate. No bacteria were recovered
Fig. 6. Matrix collagenase activity in chicken pericardial fluid and blood plasma examined using gelatin zymography. (a) Representative zymograms of pericardial fluid and blood plasma from normal chickens, chicken with sub-clinical heart disease, and ascitic chickens. The column labelled MMP STD indicates molecular markers of MMP-9 (92 kDa), pro-MMP-2 (72 kDa), and active MMP-2 (69 kDa) obtained from cell culture medium of the human fibrosarcoma HT 1080 cell line. The activity of MMPs in the pericardial fluid and plasma revealed the presence of both pro-MMP-2 (72 kDa), and active MMP-2 (69 kDa), but MMP-9 (92 kDa) was absent. (b) Trends in relative gelatinolytic activity of MMP-2, pro-enzyme, and the activated form in pericardial fluid and blood plasma from leghorns (resistant to ascites), slow growing feed restricted broilers (Broiler R, low risk of ascites), fast growing ad libitum fed broilers (Broiler A-L, high risk of ascites), and broilers with heart failure and ascites (CHF-Ascites). The values represent means (SE) from 2 leghorns, 4 slow growing broilers, 7 fast growing broilers, and 4 ascitic broilers.
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from the pericardial fluid. The study of activity of MMPs in blood plasma and pericardial fluid revealed the presence of both pro-MMP-2 (72 kDa), and active MMP-2 (69 kDa), but MMP-9 (92 kDa) was virtually absent. Fig. 6a shows typical zymograms from normal and abnormal individuals. The relative gelatinolytic activity of MMP-2, in particular the activated form, was higher (p < 0:05) in pericardial fluid from broilers (generally prone to heart failure and ascites) in comparison to leghorns (resistant to heart disease), and was the highest (all p < 0:05) in birds with fulminat heart failure and ascites (Fig. 6b). Noteworthy are relatively higher pro-MMP-2 activities in fast growing broilers (high risk of ascites) in comparison to slow growing broilers (low risk of ascites). Generally, the active form of MMP-2 was higher in PE than in blood plasma of broiler chickens.
4. Discussion From the study of Nakamura et al. (1999), our retrospective data (Olkowski et al., unpublished observations), and the present results it is evident that lesions in the pericardium occur in the vast majority (>90%) of broilers succumbing to heart failure and ascites. Based on echocardiographic observations and post-mortem data, it appears that pericardial lesions represent early changes associated with the development of heart failure and ascites in broilers, but because pericardial lesions almost always occur together with pathological changes in the myocardium, their aetiological identity it is not clear at present. Because of the close anatomical relationship with the myocardium, the lesions associated with the pericardium may have a wide range of possible adverse effects on cardiac muscle patho-physiology and heart pump performance. Therefore, the pericardial lesions described here ought to be considered in the context of their potential effects on the mechanical performance of the heart pump, as well as from the perspective of electrophysiological, functional, and biochemical characteristics. Based on echocardiagraphic examination, it is apparent that pericardial lesions are present before overt signs of heart failure appear. Notably, pericardial adhesions may affect cardiac diastolic and systolic function, whereas excess fluid in the pericardial space may increase the haemodynamic burden of the heart mainly during diastole. The restrictive effect of PE and/or pericardial adhesions therefore ought to be considered in the aetiology of heart pump failure. Poor performance of the heart pump in broilers with fulminant heart failure and ascites is clinically evidenced by impaired ventricular wall motion, reduced fractional shortening, and reduced cardiac output (Olkowski and
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Classen, 1999; Olkowski et al., 1999). All these changes are consistent with the restrictive effects of PE. Increased haemodynamic burden in heart chambers associated with excessive pressure in the pericardial space may contribute to the rise in hydrostatic pressure in the venous circulation, and therefore may be one of the more significant factors contributing to the formation of ascites in affected broilers. In comparison to other classes of chickens, fast growing broilers show a high incidence of cardiac dysrhythmia (Grashorn, 1994; Olkowski et al., 1997; Olkowski and Classen, 1998; Korte et al., 1999). The clinical course of both acute heart failure (SDS) and chronic heart failure and ascites appears to be closely associated with cardiac arrhythmias (Grashorn, 1994; Olkowski and Classen, 1997, 1998; Korte et al., 1999). The increased susceptibility of the broiler heart to arrhythmic activity may be associated with lesions in the pericardium as well as in the myocardium. There is little doubt that lesions in the myocardium may compromise electrical stability of the heart and lead to fatal arrhythmia. However, lesions of the pericardium in broilers may also increase the risk of arrhythmic activity. The issue of pericardial lesions as a potential arrhythmogenic factor in fast growing broilers deserves attention for at least two reasons: (1) because of the central role of the epicardial surfaces of the ventricles in the propagation of the depolarization wave (Kisch, 1951; Moore, 1965), and (2) cardiac irritability is higher in fast growing broilers in comparison to slower growing broilers (Greenlees et al., 1989). Injury in the pericardium may create a milieu conducive to electrophysiological disturbances in the heart and arrhythmia (Ware et al., 2000). Also, spatial/temporal non-uniformity, and the multi-focal nature of depolarization of the epicardial surfaces of the ventricles in the chicken (Kharin et al., 2001) may further increase the periods of electrical instability of the heart. This may be of importance in arrhythmogenesis as well as in the process of degeneration of arrhythmic episodes into fatal ventricular fibrillation (Olkowski and Classen, 1997). The pathological process that initiates pericardial effusion, adhesions and/or epicardial surface fibrosis is not known, but in view of the present findings may be associated with excessive activity of MMP-2. The importance of MMPs in myocardial pathological remodeling has been documented (Dollery et al., 1995; Coker et al., 1999; Peterson et al., 2001). Our previous study (Olkowski et al., 2001) indicated that the pathogenesis of heart failure and ascites in fast growing broilers is associated with the increased activity of MMP-2 in heart tissue. However, considering that in the present study higher levels of activated MMP-2 was seen in PE of ascitic birds as well as in broilers with pathological evidence of sub-clinical heart disease, it can be inferred
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that increased MMP activity is a factor that appears early in the development of heart failure and ascites. Further, it is noteworthy that the MMP-2 levels (the active form) in broilers, was higher in PE fluid than in blood plasma. Thus, the local synthesis and/or activation of MMPs may be aetiologically associated with the development of pericardial lesions, and may play a significant role in the aetiology of heart failure and ascites in broilers. Collagen degradation in soft connective tissue is mediated mainly by MMPs, with MMP-2 playing a key role in this process (Aimes and Quigley, 1995; Creemers et al., 1998). It is becoming increasingly clear that MMPs are involved in various aspects of physiological and pathological vascular remodeling (for review see Galis and Khatri, 2002). Hypothetically, the breakdown of ECM may alter the passive properties of blood vessels, which could lead to increased vascular permeability, thus facilitating PE and ascites in predisposed broilers. The reason why fast growing broilers show higher levels of MMP activity in blood plasma is unknown. One of the more plausible explanations is that excessive levels of MMP may be directly associated with the rapid growth potential of broiler chickens. It has been shown that the activation and/or synthesis of MMPs may be regulated by growth factors, cytokines and hormones (Tyagi et al., 1995; Rooprai et al., 2000). Hence, the very factors that are essential for growth may mediate excessive synthesis of MMP-2 in fast growing broiler chickens. The increased activity of MMPs in some fast growing broilers may be viewed as a factor increasing the risk of heart failure and ascites. In summary, the present study confirms the fact that pericardial lesions are involved in the natural history of cardiac failure in broiler chickens. For this reason, the pathogenesis of pericardial lesions themselves warrants thorough investigation. Better understanding of the relationship between PE, adhesions, and myocardial structural abnormalities, and their electrophysiological and haemodynamic significance may help to unravel the mechanisms involved in the development of both acute and chronic heart failure in broilers.
Acknowledgements The research presented in this paper has been supported by grants provided by the Poultry Industry Council.
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