A.A. Olkowski

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.

Role of thiamine status in sulphur induced polioencephalomalacia in sheep

The effects of excess dietary sulphur were studied in sheep supplemented and unsupplemented with thiamine. The diets contained either 0.19 per cent sulphur (LS) or 0.63 per cent sulphur (HS) in combinations with 14 mg kg-1 thiamine (LB1) or 243 mg kg-1 thiamine (HB1). A total of 56 two-month-old lambs were used. Groups consisting of nine, nine, 22 and 16 lambs were fed LS-LB1, LS-HB1, HS-LB1 and HS-HB1 diets, respectively for 14 weeks. Out of 22 lambs fed the HS-LB1 diet, seven lambs developed neurological signs between the third and eighth week of the trial. Two of these lambs died, three that were in extremis were euthanased, and two recovered completely. All clinically affected animals had extensive malacic lesions in the cerebral cortex, midbrain and brainstem. None of the lambs from the LS groups or HS-HB1 group developed clinical signs. Several clinically normal lambs from the HS-LB1 group had necrotic lesions in their brains at gross and microscopic examination. Supplementation with dietary thiamine prevented development of clinical signs, but did not totally prevent development of microscopic brain lesions. Brain thiamine concentration, transketolase activity and thiamine pyrophosphate (TPP) effect were not different (P greater than 0.05) among groups. There was a strong effect (P less than 0.0001) of dietary thiamine supplementation on blood thiamine concentration and TPP effect. Blood thiamine concentration was higher whereas TPP effect was lower in the thiamine supplemented sheep. Blood and tissue thiamine concentrations in sheep exposed to high dietary sulphur did not indicate either systemic or local thiamine deficiency per se. Increased TPP effect in sheep fed the HS-LB1 diet indicated mild to moderate metabolic thiamine deficiency. Thiamine inadequacy may be an effect of an increased requirement for thiamine in animals exposed to excess dietary sulphur.

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.

The role of oxidative stress in the development of congestive heart failure in a chicken genotype selected for rapid growth

The present study examined the possible role of reactive oxygen species in the pathogenesis of heart failure in broilers. Data were collected from three groups of birds at various risk of heart failure: Leghorn chickens (resistant to heart failure), slow-growing feed-restricted broilers (low risk of heart failure), fast-growing ad libitum fed broilers (high risk of heart failure), and broilers with congestive heart failure (CHF). In the first part of the study, basic clinical parameters and ultrastructural changes were examined in the context of lipid peroxidation of the ventricular myocardium. This was followed by the study of in vitro changes in the activity of selected cytosolic enzymes (creatine kinase and lactate dehydrogenase) and mitochondrial enzymes (pyruvate dehydrogenase and α-ketoglutarate dehydrogenase) in the presence of oxidants (hydrogen peroxide or tertiary butyl hydroperoxide). The distinctive clinical feature in the fast-growing broilers and in the broilers with CHF as compared with slow-growing broilers or Leghorn chickens was a significantly lower heart rate (P <0.05). Electron microscopy revealed marked morphological changes in myocardial mitochondria in these broilers (i.e. fast-growing broilers and broilers with CHF). The level of malondialdehyde equivalents, an indicator of lipid peroxidation subsequent to generated oxidative stress, was significantly higher (P <0.05) in ad libitum fed broilers and was highest (P <0.01) in broilers with CHF. In vitro, the presence of oxidants had a detrimental effect on creatine kinase and α-ketoglutarate dehydrogenase activity, while lactate dehydrogenase activity increased. The activity of pyruvate dehydrogenase was not altered by oxidants. Our results indicate that the deterioration of heart function in fast-growing commercial broilers in our experimental model is associated with oxidative stress leading to lipid peroxidation of cellular and mitochondrial membranes, and decreased activity of myocardial creatine kinase and α-ketoglutarate dehydrogenase enzymes critical for energy synthesis and transformation pathways.

Understanding the role of sulfur-thiamine interaction in the pathogenesis of sulfur-induced polioencephalomalacia in beef cattle.

This study examined the role of sulfur (S) in the pathogenesis of S-induced polioencephalomalacia (PEM) in beef cattle in the context of thiamine status and metabolism. Thiamine, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) status in rumen fluid, blood and brain tissue were determined in beef heifers fed 2 levels of S [low S (LS) vs. high S (HS)] at 2 forage-to-concentrate ratios (F:C). High S diet did not affect ruminal and blood thiamine status. Interestingly, however, HS diet showed increased brain thiamine levels. No gross or histopathological changes indicative of PEM were detected in the brains of the heifers. Of note, during the course of the present study, we documented an outbreak of S-induced PEM in commercial feedlot steers. Brain thiamine variables in experimental animals fed HS diet were then contrasted with brain thiamine status in PEM affected feedlot steers. Interestingly, in clinically normal animals, exposure to HS diet resulted in increased levels of both TMP and TPP in the brain tissue, in comparison to animals fed LS diet. In contrast, the PEM affected brains showed overall lower levels of thiamine phosphates. It is noteworthy that TPP levels were 36.5% lower, despite 4.9-fold higher free thiamine in PEM brains compared to normal brains. Our results indicate that high dietary S may increase the metabolic demand for TPP, and that animals incapable of maintaining requisite levels of brain TPP are at high risk to develop fulminant cerebrocortical necrosis.

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.

Toxin metabolism potential in Richardson ground squirrels (Spermophilus richardsonii) in the context of chemical control of this pest

Richardson ground squirrels (Spermophilus richardsonii) are prominent pests on the prairies of North America. Rodenticides have been used for their control for several decades, but this pest continues to thrive. In this study we considered the possibility that this apparent lack of success in controlling this pest may be associated with systemic adaptation of Richardson ground squirrels to toxins. Accordingly, we examined the hepatic cytochrome P450 (CYP) enzyme system (phase I biotransformation) in Richardson ground squirrels selected from fields where rodenticides were used (exposed) and from fields where no toxins were distributed (naïve). We found that the content and activity of individual components of the CYP system including CYP, cytochrome b5, and NADPH–CYP reductase in liver microsomes were higher in exposed Richardson ground squirrels than in naïve Richardson ground squirrels. In vitro CYP-mediated activities for generic substrates representing major groups of reactions were found to be highly variable among individuals. However, systemic activity was comparable among the naïve and exposed Richardson ground squirrels. These findings indicate that Richardson ground squirrels may readily develop resistance to toxins by enhancing the functional capacity of enzymes responsible for detoxification. Therefore, it is important to carefully monitor the efficiency of toxicological methods of Richardson ground squirrels control and to revise management strategies accordingly.

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.