Incidence of BVDV1 and BVDV2 infections in cattle submitted for necropsy in Northern Germany.

The incidence of bovine viral diarrhoea virus (BVDV) 1 and 2 infections was determined in calves, young cattle and older cattle with signs of mucosal disease (MD) submitted for necropsy to three laboratories in Northern Germany between June 2000 and May 2001. At necropsy, tonsils, retropharyngeal lymph nodes, mesenteric lymph nodes, ileal Peyer's patch and spleen were collected and examined by immunohistochemistry and virus isolation. From 311 animals examined, 30 (9.6%) were positive for BVDV. All viral isolates were typed by polymerase chain reaction after reverse transcription using species-specific primers and determined to be BVDV1. Based on the distribution of lesions and viral antigen, animals with MD, persistent infection (PI) and acute, transient infection could be distinguished. Twelve of the positive animals had characteristic signs of MD: severe diarrhoea, erosive to ulcerative lesions throughout the digestive tract and severe depletion of all lymphoid tissues. Viral antigen was present in all tissues and cell types, but particularly in depleted lymphoid follicles and altered epithelium. In seven calves, viral antigen was detectable in all tissues and cell types, but lesions were mild or missing. This is typical for PI. The remaining 11 calves most likely represent animals with acute, transient infection. Distribution of antigen was more variable, predominantly restricted to lymphoid follicles and often not seen in all tissues examined. Clinical findings were combined bronchopneumonia and enteritis. The detection of BVDV1 in young calves with pneumonia and enteritis emphasizes the importance of BVDV1 and not only BVDV2 for severe respiratory and enteric diseases of calves.

Shiga-toxigenic Escherichia coli-inoculated neonatal piglets develop kidney lesions that are comparable to those in humans with hemolytic-uremic syndrome.

Kidney lesions similar to those in humans with hemolytic-uremic syndrome were observed histologically in 82 of 122 piglets inoculated intragastrically with Shiga-toxigenic Escherichia coli but not in 29 controls. The locations of lesions matched locations where Stx-2 binding and Gb3 (globotriasylceramide receptors for Stx) were identified immunohistochemically.

Changes in distribution and numbers of CD4+ and CD8+ T-lymphocytes in lymphoid tissues and intestinal mucosa in the early phase of experimentally induced early onset mucosal disease in cattle.

Mucosal disease (MD), one sequelae of bovine virus diarrhoea virus (BVDV) infection, causes severe lesions in lymphoid tissues and mucosal surfaces. Lesions are associated with the presence of cytopathogenic (cp) BVDV and initially characterized by apoptotic cell death. The objective of this investigation was to determine if this cell death is mediated only by the cp BVDV, which is known to induce apoptosis in cell culture or if immune-mediated host reactions might also contribute. Early onset MD was experimentally induced in calves by inoculation of persistently viremic calves with a closely related cp BVDV. Calves were euthanized in the early phase of infection between days 5 and 13 post-inoculation and tissues from tonsils, lymph nodes, Peyer's patches, jejunum and colon were collected. Presence of cp BVDV antigen was correlated with distribution of lymphocyte subpopulations in consecutive cryostat sections. In the lymphoid tissues, cp BVDV antigen was predominantly found in the lymphoid follicles. The increase of infected cells with time post-inoculation was paralleled by a decrease of B-lymphocytes and an increase of CD4+ T-lymphocytes. An increased number of CD8+ T-lymphocytes was seen in progressed lesions only. In the intestinal mucosa, initially multifocal, later diffuse infection with cp BVDV was accompanied by a multifocal or diffuse increase of CD4+ T-lymphocytes, respectively. Numbers of IgA+ plasma cells and CD8+ T-lymphocytes were decreased. The common change observed in lymphoid tissues and mucosa was the increase of CD4+ T-lymphocytes in sites with lesions. This might indicate a cell-mediated immune response to the cp BVDV. Besides their helper function to other cells of the immune system, activated CD4+ T-lymphocytes might also exert cytotoxic activity, induce apoptosis in target cells via Fas/Fas ligand binding and thus contribute to the severity of tissue lesions in MD.

Leukopenia and thrombocytopenia in pigs after infection with bovine viral diarrhoea virus-2 (BVDV-2).

The aim of this study was to investigate whether bovine viral diarrhoea virus-2 (BVDV-2) is pathogenic for pigs, which organs become infected and whether or to which extent the virus is excreted into the environment. Ten pigs were observed for clinical reactions after infection with a BVDV-2 strain, that has been shown to be pathogenic in calves under experimental conditions. Samples were taken to monitor thrombocyte and leukocyte counts as well as antibody development. Post mortem examinations were performed at 7, 11 and 27 days after infection. Tissue samples were collected for virus isolation, histological and immunohistological examination. All ten pigs became infected and BVDV could be re-isolated from the lymphocytes, the plasma and different lymphatic organs. The infection passed clinically inapparent, apart from a slight increase in body temperature in some animals. Some animals developed a slight leukopenia and/or thrombocytopenia. There were no macroscopic or histological lesions observed that could specifically be related to the inoculation of BVDV-2. With respect to all parameters studied, the infection and the consequences thereof were clearly less pronounced in pigs as compared to cattle, the natural host. Our results indicate, that pigs infected with BVDV-2 might develop antibodies that cross-react in tests for antibodies against classical swine fever virus.

Saccharomyces boulardii and bacillus cereus var. Toyoi influence the morphology and the mucins of the intestine of pigs.

The mode of action of probiotics is still incompletely understood. To study the interactions between probiotic micro-organisms and the host their effects on morphology and mucins of the intestinal mucosa were investigated. Fifteen clinically healthy weaned pigs were divided into three groups and received either Saccharomyces boulardii or Bacillus cereus var. toyoi or were left untreated. Sections of duodenum, proximal and mid jejunum, ileum, caecum, and colon were examined. An increase of villus length in the small intestine and a decrease in the number of goblet cells with 2.6-sialylated mucins in the large intestine were observed in both treatment groups. There were no differences in crypt morphology, number of Ki67-positive cells, total number of goblet cells and number of goblet cells with acidic, neutral, sulphated, or 2.3-sialylated mucins between groups. The results indicate an effect of Saccharomyces boulardii and Bacillus cereus var. toyoi on the intestinal architecture of pigs.

Uptake of colostral leukocytes in the intestinal tract of newborn calves.

The role of colostral immunoglobulins for the protection of newborn calves has been studied extensively, but little is known about the importance of colostral leukocytes. To study the uptake of colostral leukocytes in the intestine of calves and to determine preferential sites for this uptake, FITC-labelled colostral cells derived from the respective dams were injected into intestinal loops with/without Peyer's patches of three male Holstein Frisian calves about 5h post natum. In adjacent loops, PBS was injected as control. Loops were excised after an exposure of 1.5-2h. FITC-labelled material and cells were detected by the direct immunoperoxidase method in paraplast sections. Twenty-five consecutive sections were evaluated from each localization. Uptake of labelled material and cells was observed in all three calves in the jejunal Peyer's patch and in two calves in the ileal Peyer's patch as well. In the jejunal Peyer's patch, labelled material and cells were present in epithelium, domes and sinuses around lymphoid follicles, whereas in the ileal Peyer's patch, they were found in the sinuses only. These findings confirm that uptake of colostral leukocytes through the intestinal barrier is possible and that the preferential route of uptake is through follicle-associated epithelium of Peyer's patches.

Escherichia coli O157:H7 causes more-severe systemic disease in suckling piglets than in colostrum-deprived neonatal piglets.

Our objective was to determine if suckling neonatal piglets are susceptible to enterohemorrhagic Escherichia coli (EHEC) O157:H7 disease. Surprisingly, EHEC O157:H7 caused more-rapid and more-severe neurological disease in suckling neonates than in those fed an artificial diet. Shiga toxin-negative O157:H7 did not cause neurological disease but colonized and caused attaching-and-effacing intestinal lesions.

Ultrastructural changes in follicles of small-intestinal aggregated lymphoid nodules in early and advanced phases of experimentally induced mucosal diseases in calves.

OBJECTIVE: To investigate ultrastructural changes in follicles of small-intestinal aggregated lymphoid nodules (Peyer's patches) of calves with early and advanced phases of experimentally induced mucosal disease (MD). ANIMALS: Twenty 2.5- to 7-month-old Holstein-Friesian calves (11 females, 9 males). PROCEDURE: MD was induced in 13 of 18 calves that were persistently viremic with bovine viral diarrhea virus (BVDV). Eight of the 13 calves were euthanatized before the onset of clinical signs of MD, and 5 were euthanatized after becoming moribund with MD. Five persistently viremic calves and 2 calves without BVDV served as controls. Specimens of small-intestinal aggregated lymphoid nodules were prepared for transmission electron microscopy. RESULTS: The ultrastructure of follicles of small-intestinal aggregated lymphoid nodules from healthy calves was consistent with that in sheep. In the early phase of MD, changes were characterized by numerous apoptotic lymphocytes and macrophages with apoptotic bodies. In more advanced lesions, affected lymphoid follicles consisted of macrophages and variable numbers of follicular dendritic cells (FDC), whereas others did not contain FDC. In moribund calves, small follicles consisting predominantly of FDC and follicles with central cavities surrounded by macrophages, and few neutrophils were observed. CONCLUSIONS AND CLINICAL RELEVANCE: The ultrastructural changes in lymphoid follicles of small-intestinal aggregated lymphoid nodules indicate apoptosis of lymphocytes as an initial event. The development of small follicles consisting predominantly of FDC or the complete loss of follicular architecture in advanced phases of MD is determined by the intensity of apoptosis of lymphocytes, the capacity of the macrophages for uptake, and the reorganization of a stromal network.

Immunohistochemical demonstration of African horse sickness viral antigen in tissues of experimentally infected equines.

African horse sickness virus (AHSV) antigen was demonstrated immunohistochemically in formalin-fixed, paraffin-embedded sections of tissues collected from three ponies suffering from the peracute form of the disease and from one pony affected by the fever form. The pattern of the antigen distribution indicated a particular organ tropism characterised by an accumulation of AHSV antigen in cardio-pulmonary tissues of the animals with the peracute disease and in the spleen of the pony with the fever form. AHSV antigen was identified in endothelial cells of small blood vessels, particularly capillaries and in large mono-nuclear cells resembling macrophages or reticular cells of lymphatic tissues. Occasional circulating mononuclear cells with the morphology of monocytes were also positively stained within the larger vessels. The immunohistochemical results confirm earlier work suggesting that AHSV may have different tropisms to particular organs during various forms of the disease and that different target cell populations exist in vivo. Immunohistochemistry may be an additional useful method for diagnostic and research purposes in AHS.

Immunohistochemical demonstration of African horse sickness viral antigen in formalin-fixed equine tissues.

The distribution of viral antigen was studied in various tissues of three ponies, aged 3-4 years, infected experimentally with a virulent strain of African horse sickness virus (AHSV) serotype 4. Tissues were collected from the animals in the terminal stage of the peracute form of the disease and from one noninfected horse, included as a control. A polyclonal antibody with specificity for AHSV, plus the nonstructural protein NS2, was used in a sensitive avidin-biotin-peroxidase-complex (ABC) method performed on formalin-fixed, paraffin-embedded tissue sections. AHSV antigen was located primarily in endothelial cells of capillaries and small venous and arteriolar vessels, particularly of cardiopulmonary tissues. Viral antigen was also identified in cells resembling macrophages and in reticular cells of spleen and lymph nodes. The pattern of viral antigen labeling in some lymph nodes along the mantle zone of lymphoid follicles was compatible with the morphology of cellular processes of follicular dendritic cells. In some tissues, viral antigen was detected occasionally in circulating cells, probably monocytes, within the larger vessels. These findings suggest that endothelial cells, and to a lesser extent mononuclear cells, are the main target cells of AHSV infection during the late stage of the peracute form of the disease.

Identification of leucocyte surface antigens in paraffin-embedded bovine tissues using a modified formalin dichromate fixation.

A modified fixative of formalin dichromate was combined with a cold embedding procedure for the preservation of bovine leucocyte surface antigens. Fourteen monoclonal antibodies recognizing seven bovine leucocyte surface antigens (BoCD1w2, BoCD4, BoCD8, BoWC1, BoWC3, BoWC4 and BoIgM) were applied as primary antisera in a sensitive avidin-biotin-peroxidase complex detection method. The staining results were compared with those obtained in cryostat and routinely formalin-fixed sections of corresponding tissue samples. Using the modified formalin dichromate fixative and the cold embedding procedure, all the leucocyte surface antigens tested were detectable immunohistologically in paraffin sections with a generally more distinct staining than in traditionally processed tissues. Morphological structures were better preserved than in cryostat sections but, to some extent, were poorer when compared with routinely formalinfixed tissues. However, this method suggests that there are only mild masking effects and provides an alternative to the use of unfixed material, particularly for morphological-immunohistochemical investigations.

Organ and tissue distribution of the antigen of the cytopathogenic bovine virus diarrhea virus in the early and advanced phase of experimental mucosal disease.

To study the development of lesions in mucosal disease, the spread of cytopathogenic (cp) bovine virus diarrhea virus (BVDV) to different organs was examined in relation to the time post inoculation (pi). Mucosal disease was induced in 15 persistently viremic cattle from two herds by intranasal inoculation with antigenically similar cp BVDV strain. This strain reacted with one additional monoclonal antibody when compared to the corresponding herd-specific non cytopathogenic (ncp) isolate. Twelve cattle were euthanized at days 3, 5, 7, 9 and 13 pi in the early phase before they developed clinical signs of mucosal disease, three in the advanced phase when they were moribund and three served as controls. Antigen of the cp BVDV strains was selectively detected in tissue sections by immunohistochemistry. In the early phase, varying amounts cp BVDV were present most consistently in tonsils, lymph nodes, Peyer's patches and lymphoid nodules in the large intestine. In the lymphoid tissues, first a few cells in single lymphoid follicles, then groups of lymphoid follicles contained antigen. In intestinal epithelium, cp BVDV antigen was found focally in the early phase of mucosal disease. Its diffuse distribution in the late phase corresponded with clinical signs of diarrhea.

Experimental mucosal disease of cattle: changes in cell proliferation in lymphoid tissues and intestinal epithelium.

In mucosal disease of cattle, the initial and most severe lesions are found in the lymphoid follicles and intestinal crypts, both sites showing a high cell proliferation rate. In the present study, the changes in the number and distribution of proliferating cells were investigated immunohistologically, by demonstrating the proliferation-associated nuclear antigen, Ki-67. Tissues were obtained from 30 cattle, all of which had a persistent natural infection with non-cytopathogenic (ncp) bovine virus diarrhoea virus (BVDV), and 22 of which were subsequently inoculated with antigenically closely related strains of cytopathogenic (cp) BVDV to produce mucosal disease (MD); the remaining eight cattle served as uninoculated controls. Twelve of the inoculated cattle were killed before the onset of clinical signs of MD ("early phase"), and 10 when they were moribund ("late phase"). In the controls, the lymphoid follicles in lymph nodes and Peyer's patches consisted predominantly of Ki-67-positive cells; high numbers of such cells were observed in the crypts of the small intestine and moderate numbers in the crypts of the large intestine. In the early phase of MD, the number of Ki-67-positive cells in the lymphoid follicles and in the domes of the Peyer's patches gradually decreased; but in the mucosa, foci of crypts with increased numbers of Ki-67-positive cells were observed. In the late phase of MD, only a few Ki-67-positive cells were present in the lymphoid follicles. There was an increase in the number of Ki-67-positive cells in most crypts in the small and large intestine, but foci of crypts without Ki-67-positive cells occurred.

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.

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.

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.