Transfer of pyrrolizidine alkaloids from various herbs to eggs and meat in laying hens.

To investigate the potential transfer of pyrrolizidine alkaloids (PAs), laying hens were fed for 14 days with diets containing 0.5% of dried common ragwort, common groundsel, narrow-leaved ragwort or viper's bugloss, or 0.1% of common heliotrope. This resulted in total PA levels in feed of respectively 5.5, 11.1, 53.1, 5.9 and 21.7 mg kg-1, with varying composition. PAs were transferred to eggs, in particular yolk, with steady-state levels of respectively 12, 21, 216, 2 and 36 µg kg-1. Overall transfer rates for the sum of PAs were estimated between 0.02% and 0.23%, depending on the type of PAs in the feed. In animals slaughtered shortly after the last exposure, levels in meat were slightly lower than those in eggs, levels in livers somewhat higher. When switched to clean feed, levels in eggs gradually decreased, but after 14 days were still above detection limits in the hens exposed to higher PA levels. Similar was the case for meat and especially kidneys and livers. It is concluded that the intake of PA containing herbs by laying hens may result in levels in eggs and meat that could be of concern for consumers, and as such should be avoided.

Optimal combinations of acute phase proteins for detecting infectious disease in pigs.

The acute phase protein (APP) response is an early systemic sign of disease, detected as substantial changes in APP serum concentrations and most disease states involving inflammatory reactions give rise to APP responses. To obtain a detailed picture of the general utility of porcine APPs to detect any disease with an inflammatory component seven porcine APPs were analysed in serum sampled at regular intervals in six different experimental challenge groups of pigs, including three bacterial (Actinobacillus pleuropneumoniae, Streptococcus suis, Mycoplasma hyosynoviae), one parasitic (Toxoplasma gondii) and one viral (porcine respiratory and reproductive syndrome virus) infection and one aseptic inflammation. Immunochemical analyses of seven APPs, four positive (C-reactive protein (CRP), haptoglobin (Hp), pig major acute phase protein (pigMAP) and serum amyloid A (SAA)) and three negative (albumin, transthyretin, and apolipoprotein A1 (apoA1)) were performed in the more than 400 serum samples constituting the serum panel. This was followed by advanced statistical treatment of the data using a multi-step procedure which included defining cut-off values and calculating detection probabilities for single APPs and for APP combinations. Combinations of APPs allowed the detection of disease more sensitively than any individual APP and the best three-protein combinations were CRP, apoA1, pigMAP and CRP, apoA1, Hp, respectively, closely followed by the two-protein combinations CRP, pigMAP and apoA1, pigMAP, respectively. For the practical use of such combinations, methodology is described for establishing individual APP threshold values, above which, for any APP in the combination, ongoing infection/inflammation is indicated.

Identification of a Bartonella species in the harbor seal (Phoca vitulina) and in seal lice (Echinophtirius horridus).

To explore whether harbor seals (Phoca vitulina) are exposed to Bartonella spp., 35 seal lice (Echinophtirius horridus) were collected from seven seals, during their rehabilitation period in the Seal Rehabilitation and Research Center at Pieterburen, The Netherlands. Forty-eight spleen samples were collected during necropsies of other harbor seals that died during rehabilitation, or had stranded dead and were brought to the Seal Rehabilitation and Research Center for postmortem investigation. Lice were grouped into six pools, and DNA was extracted from each pool and from all the seals' spleen samples. One of the six lice pools and one spleen sample were found positive by high-resolution melt, real-time PCR amplifying partial loci of the rpoB gene, and the intergenic spacer (ITS) region. The Bartonella spp. identified in the spleen and lice were found to be identical to each other. One hundred percent sequence similarity with Bartonella henselae was found in the ITS, and 97% sequence similarity with Bartonella grahamii was detected in the rpoB gene. To the best of the authors' knowledge, this is the first report describing the detection of Bartonella spp. from a seal or any other pinniped, and from seal lice, E. horridus. The 100% sequence similarity in the ITS of the Bartonella sp. identified with the zoonotic B. henselae warrants further investigation and characterization of this organism, which may be found to be of public health importance.

A combination of behavioral and physiological indicators for assessing pig welfare on the farm.

The purpose of this research was to identify pig welfare indicators that could help in recognizing stressful practices on farm. The study evaluated behavioral and physiological indicators (cortisol and negative acute phase proteins) in 2 groups of 20 female pigs 4 months old after a 48-hr transport. The first group (A) was transported at the end of May, the second (B) in June. Behavioral observations and blood collection occurred at arrival (D1) and 28 days later (D28). Compared with within-animal control samples obtained 28 days later, pigs of Group A had increased cortisol levels and decreased albumin concentrations after arrival. As demonstrated by lesion and behavior observations, the effect on cortisol and albumin was higher in Group B pigs after a tail-biting episode occurred. The study has reported no evidence of Retinol Binding Protein (RBP) in pigs. A method developed for swine RBP quantification found RBP strongly reduced in D28 samples of Group B, confirming it to be a negative protein in pigs. The suggested combination of physiological and behavioral indicators could provide useful information on the welfare state of an animal.

Cartilage canals in the medial coronoid process of young Golden Retrievers.

The aim of the study was to establish the location of cartilage canals in the medial coronoid process (MCP) of the ulna of young Golden Retrievers, a breed that is predisposed to fractures of the medial coronoid process (FMCP). To determine whether the presence of cartilage canals could be associated with the predilection site of FMCP, the right elbows of nine young Golden Retrievers (aged 4, 6, 8, 10, 13, 16, 18, 22 and 24 weeks) were dissected and, with no prior decalcification, the formaldehyde-fixed MCPs were embedded in methylmethacrylate. The entire MCPs were serially sectioned in the frontal plane from cranial to caudal and the sections (5 microm) were routinely stained. Between the ages of 5 and 10 weeks, three main cartilage canals were visible--medial, central and lateral. All originated from the periosteum of the distal parts of the MCP and ended proximally under the articular cartilage. Branches of the main canals were seen more cranially and caudally. At the age of 13 weeks, the central canal was absent, and the remaining canals showed a smaller diameter. From 16 weeks onwards, no cartilage canals were seen. No direct relationship could be established between the predilection site of FMCP (lateral part of the MCP) and the presence/absence of cartilage canals, since both medial and lateral canals disappeared at the same age. Further research is needed to elucidate the pathogenesis of FMCP.

Monitoring health by values of acute phase proteins.

A systemic acute phase reaction may develop during infection and inflammation, due to the action of peripherally liberated proinflammatory cytokines. Hepatic metabolism changes, and negative and positive acute phase proteins (APPs) can be measured in the blood: the APPs therefore represent appropriate analytes to assess health. While they are non-specific markers, their levels change with biological effects and this can be used to assess nutritional deficits and reactive processes, especially when positive and negative acute phase variables are combined in an index. Unfortunately, at present, no comprehensive, easy-to-use and cheap system is available to assess various acute phase proteins in serum or blood samples. Protein micro-array technology may satisfy this need; it will permit simultaneous analysis of numerous analytes in the same small volume sample and enable integration of information derived from systemic reactivity and nutrition with disease-specific variables. Applying such technology may help to address health problems in many countries.

Chemical typing of porcine systemic amyloid as AA-amyloid.

Systemic AA amyloidosis is frequently reported in a wide variety of domestic and wild animal species. Porcine amyloidosis is rare and the amyloid has not been typed chemically thus far. In the present study, we have extracted porcine amyloid from formalin-fixed tissue sections. By subsequent amino acid sequencing, an N-terminal fragment was obtained identifying porcine systemic amyloid as AA amyloid. The N-terminal sequence had a great homology to bovine and ovine SAA1, suggesting that pig AA amyloid is derived from the systemic isoform of SAA. It is argued that the low incidence of amyloidosis in pigs is not likely to be attributed to unique features of porcine amyloid precursor protein. Elucidation of the basis for the high apparent resistance of pigs against amyloidosis may yield important clues for treatment and prevention of amyloidosis in other species. This is the first report on chemical identification of porcine amyloid.

The pathogenesis of and susceptibility to malabsorption syndrome in broilers is associated with heterophil influx into the intestinal mucosa and epithelial apoptosis.

Malabsorption syndrome (MAS) in broilers is characterized by enteritis and reduced body weight gain. The pathogenesis of the intestinal lesions and the reasons for susceptibility differences between broiler lines are not clear. We studied the development of enteric lesions, epithelial apoptosis, and cell proliferation in relation to susceptibility. One-day-old chickens from two broiler lines were orally inoculated with intestinal homogenate derived from MAS-affected chickens. Vacuolar degeneration and apoptosis of the villous epithelium and infiltration of heterophils into the lamina propria occurred from day 1 post-inoculation. Following heterophil accumulation, at day 4 to 6 post-inoculation, there was severe apoptosis of the crypt epithelium and villous atrophy. The susceptible broilers had a significantly greater influx of heterophils and, subsequently, severe epithelial apoptosis and cystic damage to the crypts. There appeared to be a causal relationship between heterophil influx and the onset of apoptosis. Coincident with the epithelial apoptosis, MAS-affected chickens had crypt hyperproliferation and faster epithelial turnover. Heterophil infiltration and epithelial apoptosis appear to be critical in the pathogenesis of MAS. Heterophil recruitment may be a major factor in differences in susceptibility to MAS.

Mutual paracrine effects of colorectal tumour cells and stromal cells: modulation of tumour and stromal cell differentiation and extracellular matrix component production in culture.

Interactions of tumour and stromal cells influence tumour cell proliferation and differentiation, stromal cell phenotypic transdifferentiation and secretion of extracellular matrix (ECM) components. In this study, we established a monolayer and a three-dimensional cell-to-cell interaction model between canine mammary stromal cells and human colonic carcinoma cell lines (Caco-2 and HT-29) to investigate mutual paracrine effects of tumour cells and stromal cells on (i) tumour cell differentiation, (ii) production of ECM components and (iii) phenotypic transdifferentiation of stromal cells. We showed that when Caco-2 or HT-29 cells are cultured in collagen gels, they form a few small solid cell clusters with no lumina, but when cocultured with stromal cells, the tumour cells formed glandular structures with central lumina. This fibroblast-induced organization and differentiation of Caco-2 cells (not HT-29 cells) appeared to be mediated by transforming growth factor-beta (TGF-beta). Culturing of stromal cells, Caco-2 cells or HT-29 cells alone in both monolayers and gels resulted in weak tenascin-C expression in stromal cells and HT-29 cells and no expression in the Caco-2 cells. Coculturing of stromal cells with tumour cells resulted in increased tenascin-C expression in the stromal cells and HT-29 cells and induced expression of tenascin-C in the Caco-2 cells. This induction and increased expression of tenascin-C appeared to be mediated by TGF-beta. Culturing of stromal cells, Caco-2 cells or HT-29 cells alone on monolayers and in gels resulted in a weak expression of chondroitin sulfate (CS), chondroitin-6-sulfate (C-6-S) and versican in stromal cells and no expression in Caco-2 and HT-29 cells. Coculturing of stromal cells with tumour cells on monolayers and in gels resulted in increased CS, C-6-S and versican expression in stromal cells. This tumour cell-induced expression of CS, C-6-S and versican appeared to be mediated by TGF-beta and platelet-derived growth factor (PDGF). Coculturing of Caco-2 and HT-29 and stromal cells promoted the transdifferentiation of stromal cells into myofibroblasts, and this appeared to be mediated by TGF-beta. These results suggest that TGF-beta and PDGF are part of a paracrine system involved in stromal-epithelial cell interaction important in stromal cell differentiation and ECM component production.

Serum amyloid A production by chicken fibroblast-like synoviocytes.

In brown chicken with chronic inflammatory processes of the joints amyloid arthropathy easily develops. The amyloid has been shown to be of the AA type which is derived from serum amyloid A (SAA). The aim of the present study was to investigate whether fibroblast-like synoviocytes (FLS) originating from brown chicken and other chicken breeds express SAA mRNA and produce SAA protein. FLS were isolated from the knee joint synovium of healthy brown chickens, white chickens, and broilers. The absence of macrophages in FLS cultures was confirmed by assessment of the phagocytic capability and by immunohistochemistry. Additionally, cultured cells were identified by electron microscopy and immunohistochemical staining. Expression of SAA mRNA in normal and lipopolysaccharide (LPS)-stimulated cells was assessed by in situ hybridization, Northern blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), Southern blot analysis and real-time quantitative PCR. SAA protein production was analyzed by Western blotting and ELISA. SAA mRNA was detected in unstimulated FLS isolated from the three different chicken breeds and more abundantly in those stimulated with LPS. However, SAA protein production was only detected in culture medium and cell lysate of LPS-stimulated FLS. Furthermore, FLS produced SAA in a concentration-dependent manner after stimulation with different amounts of LPS. The data suggest that during infection and inflammation chicken FLS may act as a source of articular SAA. This process may enhance development of amyloid from SAA in the joint.

Histochemical accumulation of oxidative damage products is associated with Alzheimer-like pathology in the canine.

An important lesion in Alzheimer's disease (AD) patient brains is the neurofibrillary tangle (NFT). Hyperphosphorylated tau is its major component. In a former paper we described some NFT in the canine brain. During aging, moreover, advanced glycation end products (AGE) might accumulate. Glycated tau induces lipid peroxidation in vivo and tau and AGE antigens have been mentioned to co-localize in NFT. This indicates that AGE may play an important role in Alzheimer disease (AD) by oxidation of tau. The aim of the present study was to investigate amyloid, neurofibrillary tangles, Abeta precursor protein, Abeta, tau, ubiquitin, advanced glycation end products, 4-hyroxynonenal protein and lipofuscin in a series of dogs of varying ages. The results showed a significant positive correlation between age and amyloid quantity (Congo red staining), HNE staining and lipofuscin (LF), and between amyloid quantity and HNE staining and LF. Staining for AbetaPP seemed to have a tendency to increase with age, whereas staining for tau, ubiquitin and AGE each only gave limited positive results in a proportion of the older dogs. Preliminary studies including loss of cognitive capabilities in the older dogs and chemical measurement of lipofuscin-like pigment (LFP) accumulation in brain extracts revealed an increase with old age and dementia. The Congo red, HNE and LF results suggest that deposition of amyloid with aging might be associated with formation of end products of lipid peroxidation. The finding of the limited positive signals for tau, ubiquitin and AGE in some old cases might indicate that the spontaneous brain pathology of the aged dog reveals similarities to early stages observed in AD in humans especially those with Down syndrome.

Protein folding pathology in domestic animals.

Fibrillar proteins form structural elements of cells and the extracellular matrix. Pathological lesions of fibrillar microanatomical structures, or secondary fibrillar changes in globular proteins are well known. A special group concerns histologically amorphous deposits, amyloid. The major characteristics of amyloid are: apple green birefringence after Congo red staining of histological sections, and non-branching 7-10 nm thick fibrils on electron microscopy revealing a high content of cross beta pleated sheets. About 25 different types of amyloid have been characterised. In animals, AA-amyloid is the most frequent type. Other types of amyloid in animals represent: AIAPP (in cats), AApoAI, AApoAII, localised AL-amyloid, amyloid in odontogenic or mammary tumors and amyloid in the brain. In old dogs Abeta and in sheep APrPsc-amyloid can be encountered. AA-amyloidosis is a systemic disorder with a precursor in blood, acute phase serum amyloid A (SAA). In chronic inflammatory processes AA-amyloid can be deposited. A rapid crystallization of SAA to amyloid fibrils on small beta-sheeted fragments, the 'amyloid enhancing factor' (AEF), is known and the AEF has been shown to penetrate the enteric barrier. Amyloid fibrils can aggregate from various precursor proteins in vitro in particular at acidic pH and when proteolytic fragments are formed. Molecular chaperones influence this process. Tissue data point to amyloid fibrillogenesis in lysosomes and near cell surfaces. A comparison can be made of the fibrillogenesis in prion diseases and in enhanced AA-amyloidosis. In the reactive form, acute phase SAA is the supply of the precursor protein, whereas in the prion diseases, cell membrane proteins form a structural source. Abeta-amyloid in brain tissue of aged dogs showing signs of dementia forms a canine counterpart of senile dementia of the Alzheimer type (ccSDAT) in man. Misfolded proteins remain potential food hazards. Developments concerning prevention of amyloidogenesis and therapy of amyloid deposits are shortly commented.

Analysis of cDNA sequences of feline SAAs.

Feline amyloidosis is an uncommon disorder caused by deposits of amyloid in a variety of organs. Most frequently encountered types are amyloid derived of pancreatic islet amyloid polypeptide (AIAPP) in older cats and of the apolipoprotein, apo-serum amyloid A (AA) in Abyssinian/Somali (Aby) and Siamese/Oriental (Siam) cats occurring at a relatively young age. For the AA protein of the Aby, Siam and domestic shorthair cat (DSH) breed different amino acid sequences have been described. It is not yet clear, however, whether the tendency to develop AA amyloidosis in Aby and Siam is associated with specific apoSAA protein sequences and whether this is breed specific. In this study, DNA from one Siam and two DSH cats revealed on Southern blot three bands suggesting at least three genes or gene clusters. The SAA cDNAs of hepatic mRNA from three Abys, five Siams and five DSHs were amplified by RT-PCR, cloned and sequenced. Siams and Abys had limited SAA sequence variability. All five Siams, three of which were positive for amyloid, had the amyloidogenic Siam SAA and the amyloidogenic Aby SAA sequence. Two of the Abys, both with amyloid, had the amyloidogenic Aby SAA sequence. The third Aby, without amyloid, missed its amyloidogenic sequence. The SAA sequences of the DSHs found in the present preliminary survey, suggested a possible tendency for more variability, whereas the amyloidogenic Siam as well as the amyloidogenic Aby sequence were found once. Up to five different sequences were found in a single animal. All five DSHs, moreover, had a specific sequence lacking in the Siams and Abys. The present results, especially those of the Siams, favor that in addition to the occurrence of amyloid associated SAA genes other factors such as infections and inflammatory processes are involved in the development of phenotypical amyloidosis.

Canine counterpart of senile dementia of the Alzheimer type: amyloid plaques near capillaries but lack of spatial relationship with activated microglia and macrophages.

Senile plaques and cerebrovascular amyloidosis are major histopathological lesions in the brains of aged dogs. Different types of amyloid beta protein (A beta) positive plaques are known: diffuse ones and neuritic plaques. Diffuse plaques may contain membrane-bound A beta and/or small amounts of amyloid fibrils. Neuritic plaques are cored plaques with clusters of amyloid fibrils and degenerating neurities. In human amyloid plaques, a pathogenetic role for microglia cells has been described. The aim of this investigation was to study microglia cells in relationship to canine plaques and to investigate the localisation of amyloid plaques in relationship to vasculature. The lesions were studied by hematoxylin and eosin Congo red staining and immunohistochemistry with anti-A beta for plaques, with Mac 387, anti lysozyme and a series of lectins for mononuclear cells, with anti von Willebrand Factor and Lycopersicon esculentum (tomato) lectin for the endothelium of brain capillaries. Diffuse A beta-positive plaques were found in dogs of 10.8 years and older, and cored A beta-positive plaques with birefringent amyloid in Congo red-stained sections in subjects of 15 years and older. Accumulation of microglia cells in relationship to the plaques was not obvious. With anti A beta 8-17 the distribution of the plaques in the cortical layers varied. The younger dogs had primarily diffuse plaques in the deeper layers of the cortical grey matter. The older dogs showed more cored plaques than diffuse plaques which were found throughout all cortical grey matter layers. With anti A beta x-42 more plaques were found positive, especially diffuse ones, whereas staining results of anti A beta x-40 were more confined to amyloid plaques and vascular amyloid. A close spatial relationship was found between the cored plaques and capillaries.

Progressive bovine paratuberculosis is associated with local loss of CD4(+) T cells, increased frequency of gamma delta T cells, and related changes in T-cell function.

Bovine paratuberculosis is caused by the infection of young calves with Mycobacterium avium subsp. paratuberculosis, resulting in a chronic granulomatous infection of predominantly the ileum. After an incubation period of 2 to 5 years, the disease becomes progressive in some of the chronically infected, but asymptomatic cows. This results in a protein-losing enteropathy that will ultimately be fatal. A loss of cell-mediated immune responses in symptomatic animals has been described, but no information is available concerning immune reactivity in the intestine. We sought to investigate putative disease status-associated lymphocyte subset distributions and antigen-specific functional characteristics of mononuclear cells isolated from blood, gut-associated lymphoid tissue, and the intestinal walls of 22 cows in different stages of disease and in control animals. The results demonstrated a significant decrease in CD4(+) T-cell frequency and a significant increase in TcR1-N12(+) gamma delta T-cell frequency in ileum lamina propria lymphocytes of symptomatic animals compared to the asymptomatic shedders. Immunohistology revealed that there was also an absolute decrease in the number of CD4(+) T cells in sections of the lesional ileum. Our findings also indicated that both peripheral and intestinal cell-mediated responses are decreased in symptomatic animals compared to asymptomatic animals. We conclude that the decrease in cell-mediated responses is likely related to a loss of antigen-specific CD4(+) T cells, which is most prominent in the lesional ileum from symptomatic animals, thus contributing to the progressive nature of bovine paratuberculosis.

Immunological basis of differences in disease resistance in the chicken.

Genetic resistance to diseases is a multigenic trait governed mainly by the immune system and its interactions with many physiologic and environmental factors. In the adaptive immunity, T cell and B cell responses, the specific recognition of antigens and interactions between antigen presenting cells, T cells and B cells are crucial. It occurs through a network of mediator proteins such as the molecules of the major histocompatibility complex (MHC), T cell receptors, immunoglobulins and secreted proteins such as the cytokines and antibodies. The diversity of these proteins that mainly is due to an intrinsic polymorphism of the genes causes phenotypic variation in disease resistance. The well-known linkage of MHC polymorphism and Marek's disease resistance difference represents a classic model revealing immunological factors in resistance differences and diversity of mediator molecules. The molecular bases in any resistance variation to infectious pathogens are vaguely understood. This paper presents a review of the major immune mediators involved in resistance and susceptibility to infectious diseases and their functional mechanisms in the chicken. The genetic interaction of disease resistance with production traits and the environment is mentioned.