C. Wojnarowicz

Sub-clinical necrotic enteritis in broiler chickens: Novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue

The present study revealed several previously not recognized etiological details in the development of necrotic enteritis (NE) in broilers. We provide evidence that the pathological process leading to mucosal epithelium necrosis follows morphologically distinct phases commencing at the basal domain of the mucosal epithelium and then progressively invading the entire lamina propria. Initially mucosal epithelium appears normal, but as the pathological changes progress throughout the lamina propria, the adjacent enterocytes begin to show features of necrotic cell death and the necrotic process of the epithelium progresses from being focal to locally extensive. Ultra-structural examination showed that primary changes occur at the level of basal and lateral domains of the enterocytes, whereas the apical domain of enterocytes remains intact even in the face of advanced necrotic changes. This indicates that the mucosal necrosis does not result from direct damage to the mucosal epithelium. Rather, the necrotic death of enterocytes is a consequential effect of the destruction of lamina propria, the extra-cellular matrix, and intercellular junctions. The nature of these morphological changes indicates that initiation of the pathological process leading to NE involves proteolytic factors affecting the extra-cellular matrix and cellular junctions. Further studies revealed that, indeed, the elevated activity of collagenolytic enzymes in the mucosal milieu and in intestinal tissue represents an integral component of the pathological process leading to NE. In the first instance we discovered that Clostridium perfringens strains isolated from field cases of NE secrete several potent collagenolytic enzymes. In the second instance we observed that, in comparison to controls, broilers challenged with C. perfringens isolated from field cases of NE show high levels of several collagenolytic enzymes in the intestinal tissue. A major component of the overall collagenolytic activity detected in the intestinal tissue was identified by zymography as matrix metalloproteinases (MMPs). Dominant activity was associated with MMP-2. We confirmed using immuno-histochemistry that this enzyme is expressed at high levels in mucosal tissue showing signs of NE. The high levels of collagenolytic activities, in particular associated with MMP-2, demonstrated in our studies are consistent with the nature of morphological changes observed primarily in extra-cellular matrix (ECM) at the basal domain of enterocytes, as well lateral domains of enterocytes. The lack of changes at the level of apical domain of mucosal epithelium indicates that the lipolytic aspect of alpha toxin in NE is not an essential factor in primary lesions development. Taken together, our findings indicate that the early lesions leading to NE are associated with virulence factors that induce proteolytic activity, rather than lipolytic activity.

Effects of dietary vitamin E and C supplementation on heart failure in fast growing commercial broiler chickens.

1. It has recently been shown that oxidative stress is involved in the pathogenesis of congestive heart failure (CHF) in broiler chickens. Vitamins E and C, common antioxidants, have been advocated for the prevention of heart failure in humans. The present study examines the effects of supplementation of these vitamins on incidence of CHF and prevention of oxidative stress in the myocardium. 2. Commercial male broilers were randomly allocated to three experimental groups and, respectively, offered commercial broiler diet (control), commercial diets fortified with vitamin E (960 IU/kg) or vitamin C (400 mg/kg). The broilers were monitored daily for overt signs of heart failure and clinical data including ECG and blood gas analysis were collected periodically. Lipid peroxidation was measured in cardiac tissues from apparently normal broilers and broilers developing CHF in each group using thiobarbituric acid reactive substances (TBARS) assay. 3. Overall, the incidence of CHF in broilers given diets fortified with vitamin E or vitamin C was not significantly different as compared to the control group. The incidence of overt signs of hypoxaemia was lower in the vitamin C group than in the control group. Lipid peroxidation was highest in broilers that developed CHF as compared to apparently normal broilers fed either vitamin E or C fortified diets. Neither vitamin E nor vitamin C was effective in preventing oxidative damage in broilers that developed CHF. 4. In conclusion, the present study confirmed that oxidative stress is involved in the pathogenesis of heart failure in broilers, but dietary supplementation of antioxidant vitamins did not prevent oxidative damage in broilers that developed CHF. Beneficial effects of vitamin C supplementation were evidenced by lower incidence of hypoxaemia, and the tendency to reduce the susceptibility of broilers to heart failure. However, vitamin E did not have any impact on clinical status or the incidence of CHF.

Biochemical and physiological weaknesses associated with the pathogenesis of femoral bone degeneration in broiler chickens

Femoral bone degeneration has been recognized as an important cause of lameness in broiler chickens for many years, but the pathogenesis of this condition has not been completely elucidated. The current work presents comprehensive analyses of changes associated with femoral bone degeneration based on findings from gross pathology, histopathology, biochemistry, and synchrotron-based imaging techniques. Gross lesions were predominantly seen in epiphysis and metaphysis of the proximal femur, and infrequently in distal femur, but we did not observe gross lesions in the diaphysis. Bone fractures were observed occasionally, but the most common lesions involved separation of articular cartilage of the femoral bone head, with progressive erosions of the subchondral bone. In advanced cases, on histopathological examination, changes in femoral bone were indicative of chondronecrosis and osteonecrosis. Computed tomography revealed that the degenerative process involves loss of trabecular bone. The course of the lesion development in the mineralized matrix appears to be coupled with increased bone resorption associated with excessive proliferation of pathologically altered osteoclasts. Light microscopy, Fourier transform infrared spectroscopy, and biochemical analysis provided consistent evidence that lowered protein content of the bone organic matrix is an integral component of femoral bone pathology, but these changes do not appear to be associated with excessive activity of matrix metalloproteinases. Taken together, our findings indicate that femoral bone degeneration is associated with structural changes occurring in both inorganic and organic matrix of the bone, but insufficiency in protein metabolism is most probably a primary aetiological factor in the natural history of femoral bone degeneration. However, it is important to stress that our findings do not negate the importance of bacterial infection in the evolution of this condition. Pathogens play a critical role in the progressive pathogenesis of this condition, which ultimately is manifested, in most instances, as femoral head necrosis.

Lesions of the pericardium and their significance in the aetiology of heart failure in broiler chickens.

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.

Vascular remodeling and its role in the pathogenesis of ascites in fast growing commercial broilers

This study examined the putative role of blood vessel pathology in the development of ascites in broilers. Major blood vessels (aorta, brachiocephalic arteries, pulmonary arteries, and vena cava) from normal commercial male broiler chickens, and broilers that developed congestive heart failure (CHF) with or without ascites were subjected to gross and microscopic examination. On cross-section, grossly, the arteries from normal broilers and those showing dilated cardiomyopathy without ascites appeared circular, with firm wall tone characteristic of the normal artery. In contrast, the arteries from ascitic broilers appeared flaccid and lacked elasticity, which was evidenced by collapsing, ellipsoid cross-sectional arterial lumen owing to the structural weakness of the arterial walls. Microscopically, ascitic broilers showed thinning or occasionally total loss of elastic elements in the arterial wall, and reduced network density of the structural matrix of the vascular wall, as well as increased thickness of fibers in vena cava. The structural changes seen in the major arteries from ascitic broilers are maladaptive, and as such would definitively impose an increased hemodynamic burden on the already failing heart pump. The changes in veins are indicative of pathological remodeling conducive to increased permeability of the vascular wall, particularly in the situation when a poorly distensible structure is further subjected to wall stress associated with increased pressure and volume overload. Taken together, increased hemodynamic burden and reduced structural density of the venous wall constitute conditions conducive for seepage and accumulation of ascitic fluid.

Putative cardiotoxic compounds extracted from meat meal as a potential risk factor for the development of heart failure in fast-growing commercial broilers

Thermal processing of meat products generates cardiotoxic compounds capable of inducing heart failure in both humans and laboratory animals. Such compounds may be present in broiler diets because supplements such as meat meal (MM), which are commonly used in broiler rations, are rendered at high temperature. Our objective was to evaluate whether putative cardiotoxic compounds in MM increase the risk of heart failure in broilers. The treatment and control diets were prepared by mixing the condensed MM extract (equivalent to dietary MM inclusion of 25%) or placebo (condensed extraction medium) with commercial broiler feed, and the respective diets were offered to commercial male broilers randomly allocated to either treatment or control groups. Broilers fed a diet spiked with MM extract showed a higher incidence (P<0.05) of chronic heart failure (65.5%) in comparison with the control group (55.4%). Postmortem examination upon termination of the experiment revealed that, in comparison with control broilers, broilers fed diet containing MM extract showed higher incidence of lesions indicative of subclinical heart disease evidenced grossly by ventricular dilation and pericardial effusions, microscopically by changes characteristic of cardiomyocyte degeneration, and ultrastructurally by changes in contractile elements and in mitochondria. Measurements of cardiac high-energy phosphates revealed that broilers fed the diet containing MM extract had lower (P<0.05) levels of cardiac energy reserve as compared with birds fed control diet. We conclude that cardiotoxic factors that can induce patho-physiological changes in the heart are present in MM.

Changes in eggshell structure and predisposition of broilers to health problems: is there a common pathophysiology?

Abstract A study of broiler breeder eggs differing in eggshell matrix optical density was conducted to determine the association of eggshell structural quality and the risk of disease in broilers. A total of 10 000 eggs from a broiler breeder flock were examined according to the pre-established criteria, and allocated to groups classified as having a high or low density shell matrix. The eggs from respective groups were incubated and hatched in a commercial hatchery. Samples of unhatched eggs from each group were subjected to detailed examination to establish the cause of reproductive failure. First, quality chicks from each group were raised as separate flocks in a commercial broiler barn. Group performance, morbidity, and mortality were monitored throughout the growth period. All birds were processed in a commercial plant, and condemnation data were compiled. There were significantly more unhatched eggs, and fewer quality chicks in the group classified as having a low density eggshell matrix, in comparison to the high-density group. Embryo pathology accounted for a large proportion of the overall reproductive failure in both groups, with a large proportion of embryos showing anatomical anomalies. The eggs and embryos from the low-density eggshell matrix group were three times more likely to be infected. Significantly higher production losses associated with mortality/morbidity, and condemnations of carcasses at processing were observed in broilers from the low-density eggshell group compared with the high-density eggshell group. It is concluded that common metabolic/physiological changes in breeder hens associated with eggshell pathology may be also risk factors linked with predisposition of broiler chicks to some health problems.