Variation of the intercalating proline in artificial peptides mimicking the DNA binding and bending IHF protein.

The integration host factor (IHF) is a protein which sequence specifically induces a bend of double-stranded DNA by more than 160°. Based on IHF as lead structure, a peptide mimic was introduced resembling the positively charged body of the protein by a lysine dendrimer and the minor groove recognition loop by a cyclopeptide. The proline located close to the tip of the recognition loop intercalates between the base pair plane. It was modified in order to evaluate the influence of the side chain residue with respect to size (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), aromaticity (phenylalanine), conformation of the five-membered ring [(4R)-fluoroproline, (4S)-fluoroproline, 3,4-dehydroproline], and the peptide backbone conformation (α-methylproline) on binding dsDNA and bending the double strand. Binding and bending studies were carried out by fluorescence resonance energy transfer experiments and gel electrophoresis using DNA sequences prepared by PCR with the IHF binding site in central or terminal position. Whereas aromatic residues and α-methylproline were not tolerated as proline substitute, incorporation of (4S)-fluoroproline and 3,4-dehydroproline provided enhanced binding.

The development of early vs. late onset mucosal disease is a consequence of two different pathogenic mechanisms.

Bovine viral diarrhea (BVD) virus is the causative agent of fatal mucosal disease (MD) of cattle. Experimental induction of MD can be achieved by superinfection of calves persistently viremic with a noncytopathic (ncp) BVD virus using an antigenically similar cytopathic (cp) BVD virus. Here we describe the characterisation of BVD viruses isolated from three cases of experimentally induced MD. One animal developed clinical symptoms two weeks after superinfection (early onset MD), while the onset of disease in the other two cases occurred with a delay of months (late onset MD). Antigenic characterisation of the viruses was performed using a panel of monoclonal antibodies against the E2 glycoprotein. For genetic analysis, RT-PCR was applied to amplify specific insertions and duplications in the NS2-3 genomic region of the cp BVD viruses. In addition, these amplicons and fragments of the viral E2 genes were sequenced. The results showed that in the case of early onset MD the cp BVD virus isolated after begin of disease was identical to the one used for superinfection. In contrast, the cp BVD viruses isolated from the two animals with late onset MD were obviously the result of genetic recombinations between the persistent ncp and the superinfecting cp BVD viruses. We conclude that early and late onset MD are the consequence of different pathogenic mechanisms.

Experimental mucosal disease in cattle: changes in the number of lymphocytes and plasma cells in the mucosa of the small and large intestine.

Changes in the number of lymphocyte and plasma cell subtypes were investigated in the lamina propria and in the epithelium of the small and large intestine of cattle with mucosal disease. Mucosal disease had been induced experimentally in seven out of 13 animals persistently viremic with non cytopathogenic BVD-virus by inoculation with a matching cytopathogenic BVD-virus. For comparison, six clinically healthy, persistently viremic cattle were used. IgA+, IgM+ and IgG1+ plasma cells, BoCD4+, BoCD8+ and gamma delta + T-lymphocytes, and the antigen of the cytopathogenic BVD-virus were demonstrated in tissue sections by immunohistochemistry. Distribution of cellular subtypes in the controls was consistent with data reported from non infected cattle. In cattle with mucosal disease, a decrease in the number of plasma cells which was significant for IgA+ and IgM+, but not for IgG1+ plasma cells was found in the lamina propria. The number of BoCD4+ T-lymphocytes was reduced in the small intestine, whereas their number per mm2 of mucosa was increased in the large intestine. Numbers of intraepithelial BoCD8+ and gamma delta + T-lymphocytes were severely decreased. Antigen of the cytopathogenic BVD-virus was detected predominantly in epithelial cells of the crypts. Overall there is a severe loss of effector cells which are essential components of the humoral and cell mediated immune protection of the mucosal barrier. The decrease of immunoregulatory cells in the lamina propria and epithelium may contribute to the transformation of mucosal architecture in mucosal disease.

Response of the germfree rat colonic mucosa to dietary endotoxins.

Suggestive to induce an immunoreactive response in the colon mucosa of germfree rats 3 diets, autoclaved or gamma-irradiated, respectively, were administered to groups of 30 days old GF Ztm:SPRD rats. Diets and rats were studied in parallel after a feeding period of 20 days. The microbiological controls confirmed the sterility of diets and animals. The aqueous suspensions of one diet displayed obviously dead yeast and Gram positive and negative bacteria, which consistently were evident in colonic cast preparations of animals fed with this diet. These findings apparently were not related to the pathohistological alterations, observed in hematoxilin-eosin stained colon sections. However, dietary endotoxin concentrations between 0.6 and 10 micrograms LPS/g corresponded with the endotoxin concentrations in feces (0.3 to 3.1 micrograms LPS/g wet weight) and in colonic tissue (0.01 to 0.6 microgram LPS/g wet weight). These endotoxins obviously mediated a dose-dependent immunoreactive response of the colonic mucosa: in parallel to the dietary endotoxin content, the cellular infiltrations ranged from single mononuclear cells to severe, cell mediated, mucosal alteration. GF rats, used as an experimental animal model for inflammatory disorders of the intestine obviously necessitate diets with low endotoxin concentrations.

Experimental mucosal disease in cattle: changes of lymphocyte subpopulations in Peyer's patches and in lymphoid nodules of large intestine.

Changes in the number and distribution of lymphocyte subtypes were investigated in Peyer's patches in the jejunum and ileum, and mucosa-associated lymphoid nodules in the proximal colon and rectum of cattle with end-stage mucosal disease. Mucosal disease had been induced experimentally in seven of 13 animals by inoculation with cytopathogenic bovine viral diarrhea virus (cp BVD-virus). For comparison, six clinically healthy, persistently viremic cattle were used. IgM+, IgA+, BoCD4+, BoCD8+ and gamma delta TCR+lymphocytes, and the cp BVD-viral antigen were visualized in tissue sections by immunohistochemistry. In cattle with mucosal disease, the size of lymphoid follicles was significantly decreased in all localizations resulting in decreased numbers of B-lymphocytes per average follicular area. In most animals domes were missing and epithelium was invaginated into the lymphoid follicles. Numbers of BoCD4+ and BoCD8 + T-lymphocytes were increased per mm2 of lymphoid follicle. Conversion of these counts into number of cells per average follicular area revealed, however, that the absolute number of BoCD4 + T-lymphocytes had decreased within lymphoid follicles and there was no distinct change of BoCD8 + T-lymphocytes in comparison to the controls. Interfollicular areas were less densely populated due to reduced numbers of BoCD4 + and BoCD8 + T-lymphocytes. cp BVD-viral antigen was detected predominantly in epithelial cells and in cells with dendritic morphology within lymphoid follicles. This may indicate that the severe depletion of B-lymphocytes in the lymphoid follicles is due to alterations of the microenvironment. The decrease of BoCD4 + and BoCD8 + T-lymphocytes does not support the hypothesis of T-cell-mediated tissue damage. Destruction of mucosa-associated lymphoid nodules does not only lead to local disruption of the gastrointestinal barrier, but will reduce the seeding of effector cells to the mucosa and therefore impair the defense mechanisms of the gastrointestinal barrier.

Ultrastructural study of the uptake of ferritin by M cells in the follicle-associated epithelium in the small and large intestines of pigs.

Uptake of ferritin by M cells in follicle-associated epithelium at various sites in the small and large intestines was examined in 4 healthy 5-week-old pigs by use of electron microscopy. A 2.5% solution of ferritin in saline was injected into ligated loops of the jejunum and ileum containing aggregations of lymphoid follicles (Peyer's patches), as well as into intestinal loops containing lymphoglandular complexes at the ileocecal junction, in the central colonic flexure, and in the rectum. As negative control, saline solution was injected into loops at identical localizations. After an exposure period of 2 hours, uptake of ferritin by M cells, but not by enteroabsorptive cells of the small and large intestines, was observed. Numbers of M cells with ferritin and total M cells were counted and the percentage was calculated. Total number of M cells was highest in lymphoglandular complexes in the rectum and lowest on domes of the ileal Peyer's patch. High numbers of M cells with ferritin were found on domes of the jejunal Peyer's patch, and in lymphoglandular complexes at the ileocecal entrance and in the rectum. Only a few M cells on domes of the ileal Peyer's patch and in lymphoglandular complexes in the central colonic flexure contained ferritin. The percentage of M cells with internalized ferritin was similar on domes of the ileal Peyer's patch, and in lymphoglandular complexes at the ileocecal junction and in the rectum. It was higher on domes of the jejunal Peyer's patches and lower in lymphoglandular complexes of the central colonic flexure. Ferritin was found in the apical tubulovesicular system, multivesicular bodies, and a few vacuoles in the central area of M cells. Ferritin was exocytosed into the lateral intercellular spaces next to M cells. Uptake of ferritin by intraepithelial cells in the follicle-associated epithelium could not be documented, but ferritin was present in vesicles of subepithelial macrophages.

[The hemorrhagic form of acute bovine virus diarrhea: literature review and case report]. / Die hämorrhagische Verlaufsform der akuten Bovinen Virusdiarrhö: Literaturübersicht und Fallbericht.

Infection of cattle with certain strains of BVD-virus causes a severe thrombocytopenia. The most obvious clinical and pathological lesions are multiple hemorrhages. Until now, problems with the Hemorrhagic syndrome have been reported predominantly from veal calf operations in the U.S.A. This publication presents first a literature review about the Hemorrhagic syndrome. The current data are based upon retrospective studies of field cases and experimental infections. Afterwards clinical, pathological and virological findings from three herds of veal calves in Germany are reported in which hemorrhagic diathesis was observed in calves and BVD-virus was isolated from tissues of diseased calves. The findings in these herds closely resemble the ones described in the literature for the Hemorrhagic syndrome. Therefore, infection with BVD-virus should be considered as differential diagnoses in Germany too, when hemorrhagic diathesis is observed in cattle.

Lymphocyte subpopulations in jejunal and ileal Peyer's patches of calves with experimental Salmonella dublin infection.

Changes of lymphocyte subpopulations in jejunal and ileal Peyer's patches (JPP and IPP) of six calves inoculated with Salmonella dublin were investigated at 9 hours, 1, 2, 3 and 7 days post inoculation (p.i.) using immunohistochemistry. Reactive areas and area percentages of B-lymphocytes, as well as CD4+, CD8+ and gamma delta T-lymphocytes within the different compartments of PP were estimated using computer-assisted morphometric analysis. A significant, linear decline of the areas of lymphoid follicles and domes in JPP and IPP due to depletion of B-lymphocytes was found. The rate of decline was similar in JPP and IPP, but more severe in lymphoid follicles than in domes. Intraepithelial cells in follicle-associated epithelium changed from predominantly B-lymphocytes in controls to CD8+ T-lymphocytes in inoculated calves and clusters of B-lymphocytes were observed above domes at days 1 and 2 p.i. Areas of CD4+ and CD8+ T-lymphocytes within lymphoid follicles and domes were increased at 3 and 7 days. p.i. resulting in decreased compartmentalization of the normally segregated T- and B-lymphocyte populations. The increase of CD4+ and CD8+ T-lymphocytes was, however, significant in lymphoid follicles in the JPP only. No significant changes in the amount and distribution of gamma delta T-lymphocytes were observed.

Prolonged persistence of cytopathogenic bovine viral diarrhea virus (BVDV) in a persistently viremic cattle.

A bull persistently viremic with noncytopathogenic (ncp) BVDV was inoculated with the cytopathic (cp) BVDV strain TGAC, which had been found to be antigenically different from the endogenous ncpBVDV (ncpW8). Neutralizing antibodies against strains NADL and TGAC were detectable 12 days and four weeks post infection, respectively. The animal developed fever and diarrhea 15 weeks post infection. On days 3 and 8 after onset of diarrhea a cpBVDV (cpX) was isolated from feces. Antigenic analysis using monoclonal antibodies (MoAbs) showed that cpX and the endogenous ncpBVDV (ncpW8) had identical reactivity patterns except for one epitope that was neither expressed on TGAC nor on ncpW8. Using polymerase chain reaction analysis it was shown that both TGAC and cpX contained a p8 phi gene duplication combined with genomic insertions of identical size. Restriction enzyme analysis of the TGAC and cpX amplicons using four enzymes showed an identical cleavage pattern, except for HaeIII digestion where an additional fragment was observed with cpX. These results suggest that cpBVDV strain TGAC persisted in the viremic animal and apparently caused disease after 15 weeks.

Distribution of cytopathogenic and noncytopathogenic bovine virus diarrhea virus in tissues from a calf with experimentally induced mucosal disease using antigenic and genetic markers.

A comparative analysis of the distribution of cytopathogenic (cp) and noncytopathogenic (ncp) bovine virus diarrhea disease (BVD) virus in tissues from a calf with experimentally induced mucosal disease was performed using immunohistology and polymerase chain reaction after reverse transcription (RT-PCR) of viral RNA. For immunohistology, an antigenic marker on the superinfecting cp BVD virus defined by a monoclonal antibody (mab) was used, and overall presence of antigen was assessed with a pestivirus specific mab. The primers selected for RT-PCR detected the genomic insertion in the p125 region of the superinfecting cp BVD virus. Both methods gave consistent results.

[The significance of bredavirus as a diarrhea agent in calf herds in Lower Saxony]. / Zur Bedeutung des Bredavirus als Durchfallerreger in niedersächsischen Kälberbeständen.

The objective of this investigation was to determine the distribution of Bredavirus in cattle herds in Lower Saxony and to evaluate its significance as potential cause of diarrhea in calves. Fecal samples and paired blood samples of 119 diarrheic and 46 healthy calves up to two months of age were collected from herds where diarrhea of calves was a problem. Fecal samples were examined for Breda-, rota- and coronavirus by solid phase immune electron microscopy and by ELISA, for K99-positive E. coli and salmonella by microbiological methods, and for cryptosporidia in smears. Antibody titers against Bredavirus, total serum protein and serum gamma globulin content were evaluated in the blood samples. Bredavirus was found in fecal samples from 5% (n = 6) of diarrheic calves which came from four different herds, but not in healthy calves. Rotavirus (31.9%), coronavirus (18.5%) and cryptosporidia (29.9%) were detected more frequently in fecal samples than Bredavirus. In this investigation rotavirus, coronavirus and cryptosporidia were present in addition in all herds where Bredavirus was found. In contrast to the low percentage of fecal samples containing Bredavirus, antibody titers in 75% of calves confirmed the high prevalence of Bredavirus infection in the cattle population of Lower Saxony.

Uptake of ferritin by follicle-associated epithelium in the colon of calves.

Uptake of macromolecules (e.g., ferritin) by M cells in follicle-associated epithelium in small and large intestine was investigated in three healthy, conventionally raised, 2- to 3-week-old, female Holstein Frisian calves. A 2.5% solution of ferritin was injected into the ligated loops in mid-jejunum, in terminal ileum, in the ascending colon adjacent to the ileocecal junction, and in the proximal loop of the ascending colon containing gut-associated lymphoid tissue. After exposure times that ranged from 82 to 165 minutes, ferritin was detected in M cells of domes in the small intestine, as well as in cells in follicle-associated epithelium of proprial lymphoid nodules and lymphoglandular complexes of colon that morphologically resembled M cells of small intestine. Ferritin was found in apical invaginations, apical vesicles, multivesicular bodies, basal vesicles, and adjacent intercellular spaces. In addition to ferritin, apical vesicles, multivesicular bodies, and intercellular spaces contained 50-nm membrane-bound particles. More ferritin was endocytosed by M cells of the small intestine than by M cells of the large intestine. In the large intestine, higher amounts of ferritin were found in M cells of follicle-associated epithelium overlying proprial lymphoid nodules than in M cells of follicle-associated epithelium in the depth of lymphoglandular complexes. Based on these results, we concluded that M cells of follicle-associated epithelium in the colon of calves provide a route for antigen uptake into the intestinal lymphoid system.

M cells in the rectum of calves.

The morphology of gut-associated lymphoid tissue and the ultrastructure of overlying lymphoepithelium of newborn and three-week-old conventionally raised calves were compared. In all calves patches of lymphoid nodules were found in the terminal rectum. In newborn calves lymphoid nodules in the submucosa with caps of lymphoid tissue in the lamina propria predominated. In three-week-old calves lymphoglandular complexes were as numerous as lymphoid nodules with caps. Scanning and transmission electron microscopical examination of superficial lymphoepithelium over caps and lymphoepithelium in epithelial diverticula of lymphoglandular complexes revealed groups or single cells morphologically resembling M cells, but with widely varying apical processes. To investigate whether these putative M cells in rectal lymphoepithelium internalise and transport macromolecules across the epithelial barrier, ferritin was injected into the rectum of three-week-old calves. Eighty to 150 minutes after exposure ferritin was detected in cells resembling M cells. Thus these cells ought to be considered as M cells. It may be hypothesised that gut-associated lymphoid tissue with specialised lymphoepithelium in the rectum of calves provides a route for the uptake of antigen.

Distribution of antigen of noncytopathogenic and cytopathogenic bovine virus diarrhea virus biotypes in the intestinal tract of calves following experimental production of mucosal disease.

Mucosal disease can be experimentally induced by inoculating calves persistently viremic with noncytopathogenic (ncp) Bovine Virus Diarrhea Virus (BVDV) with an antigenetically closely related cytopathogenic (cp) BVDV strain. Calves suffering from mucosal disease develop severe intestinal lesions causing breakdown of the gastrointestinal barrier and death. Knowledge about tissue distribution of ncp/cp biotypes of BVDV may contribute to the understanding of the pathogenesis of these lesions. Distribution of cpBVDV versus ncpBVDV was demonstrated in the intestinal tract of nine calves with experimentally induced mucosal disease and in five persistently viremic calves. Biotypes were distinguished immunohistochemically in organ tissues using monoclonal antibodies against marker epitopes on the viral surface glycoprotein gp53. In persistently viremic calves ncpBVDV was present in a few epithelial cells, mononuclear cells and intramural ganglia. A multifocal reaction was observed in vascular walls. In calves with mucosal disease a striking increase of antigen containing cells occurred. Viral antigen in these cells reacted with marker antibodies for cpBVDV. A distinct tissue distribution of biotypes was observed in intramural ganglia and duodenal glands. Severe tissue damage was correlated to the presence of cpBVDV antigen. This indicates the importance of cpBVDV for the development of lesions. Interactions of cpBVDV and immunemediated mechanisms will need further investigation.

[Animal nutrition for veterinarians--recent cases of clinical disorders in horses after intake of ionophore-containing feed]. / Tierernährung für Tierärzte--aktuelle Fälle Klinische Störungen bei Pferden nach Aufnahme Ionophore-haltigen Mischfutters.

Anamnesis and clinical signs of horses form five different stables after ingestion of ionophores are reported and techniques of feed examination are described. Within a few hours or days after feeding of new types or batches of concentrates horses fell ill. They showed colic-like symptoms with intense sweating and ataxia. Most of the sick animals died within a short time span. Samples of the concentrates were analysed and different types and amounts of ionophores were detected. In four cases contamination by monensin in concentrations of less than 5 mg to 679 mg/kg feed were found. One feed sample contained monensin (8.8 mg/kg feed) as well as salinomycin (67.3 mg/kg feed). In one case lasalocid (7.9 mg/kg feed) was present. One horse from the stable where animals had obtained concentrates containing monensin (679 mg/kg feed) was necropsied. Typical signs of monensin intoxication with severe myocardial degeneration were found. Veterinarians should be alert to this rare but severe intoxication of horses.

Reproduction of mucosal disease with cytopathogenic bovine viral diarrhoea virus selected in vitro.

Isolates of non-cytopathogenic bovine viral diarrhoea (BVD) virus from 18 persistently infected calves from one herd were compared by using monoclonal antibodies directed against the major viral glycoprotein gp53. All the isolates displayed an almost identical reaction pattern. Based on this antigenic analysis three cytopathogenic BVD and three non-cytopathogenic BVD viruses closely related to the non-cytopathogenic BVD herd isolate were selected. Six of the persistently infected calves were inoculated with a pool of the three closely related cytopathogenic BVD viruses and two with a pool of the three non-cytopathogenic BVD viruses. In addition three animals were infected with one closely related cytopathogenic BVD strain (Indiana) and two animals with the antigenetically different cytopathogenic BVD viral strain A1138/69. Regardless of the inoculation route all the animals superinfected with closely related cytopathogenic BVD viruses developed the characteristic lesions of mucosal disease within 14 days of infection. Animals which were inoculated with non-cytopathogenic BVD viruses which closely resembled the herd isolate, or with cytopathogenic BVD viruses which did not resemble the herd isolate did not develop any signs of disease. However, the latter group produced antibodies to the superinfecting virus.

[Micronema deletrix as the cause of a granulomatous nephritis in a horse]. / Micronema deletrix als Ursache einer granulomatösen Nephritis bei einem Pferd.

Infections with Micronema deletrix are described sporadically in humans and horses. This case report describes the infection with Micronema deletrix in a horse. The animal was sent to the clinic because of recurrent colic. On rectal palpation a mass was detected in the area of the right kidney and the horse was destroyed on its owners request. At slaughter a greatly enlarged right kidney interspersed with numerous white nodules was found. Histological examination revealed a granulomatous nephritis with numerous sections of nematodes. Based on their morphology and size, they were identified as Micronema.

Gut-associated lymphoid tissue in the large intestine of calves. I. Distribution and histology.

Gut-associated lymphoid tissue (GALT) in the large intestine was characterized in 12 calves (10 to 84 days old) obtained at necropsy (7, group A) or healthy animals (5, group B). Patches of mucosal lymphoid follicles were in all calves at ileocecal entrances (ICE), 23-42 cm distal to the ICE in the proximal loop of the ascending colon (proximal colon [PC] patch), and in the terminal rectum. PC patches varied from 8 to 30 cm in length. Solitary lymphoid follicles were found in the cecum of three calves, between the ileocecal entrances and the PC patch in four calves, adjacent to the PC patch in all calves, and in the ampulla recti. GALT occupied 7.8% of the large intestinal wall in animals of group A: 0.6% at the ileocecal entrance, 4.8% in the proximal colon, and 2.4% in the rectum. There were two different types of mucosal lymphoid follicles in group B: propria nodules with lymphoid follicles predominantly in the lamina propria, and lymphoglandular complexes with lymphoid follicles in the submucosa. In three 3-, 6-, and 7-day-old, germfree calves, distinct follicle-associated epithelium covered propria nodules and covering folds in depths of the lymphoglandular complexes; it was characterized by numerous intraepithelial cells and lack of goblet cells.

Gut-associated lymphoid tissue in the large intestine of calves. II. Electron microscopy.

Scanning electron microscopy of lymphoid tissue in the large intestine of three germfree calves (age 3, 6, and 7 days) revealed two different units: propria nodules and lymphoglandular complexes (LGC). Propria nodules had lymphoid tissue predominantly in lamina propria and were covered by distinct follicle-associated epithelium which lacked goblet cells; nodules were surrounded by wide crypts, which were also lined by follicle-associated epithelium towards the luminal side. Lymphoglandular complexes had lymphoid follicles in the tunica submucosa; epithelial diverticulae extended through the muscularis mucosae branching into the lymphoid nodule. In centers of lymphoglandular complexes, protrusions of lymphoid tissue were covered with distinct follicle-associated epithelium. By transmission electron microscopy cells compatible with M cells in the small intestine of calves and cells with characteristics of both enteroabsorptive and M cells were found. Follicle-associated epithelium of propria nodules and lymphoglandular complexes differed only in the relative frequency of cell types.