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Weaned 3- to 4-month-old calves were fasted for 48 h, inoculated with 10(10) CFU of Shiga toxin-positive Escherichia coli (STEC) O157:H7 strain 86-24 (STEC O157) or STEC O91:H21 strain B2F1 (STEC O91), Shiga toxin-negative E. coli O157:H7 strain 87-23 (Stx(-) O157), or a nonpathogenic control E. coli strain, necropsied 4 days postinoculation, and examined bacteriologically and histologically. Some calves were treated with dexamethasone (DEX) for 5 days (3 days before, on the day of, and 1 day after inoculation). STEC O157 bacteria were recovered from feces, intestines, or gall bladders of 74% (40/55) of calves 4 days after they were inoculated with STEC O157. Colon and cecum were sites from which inoculum-type bacteria were most often recovered. Histologic lesions of attaching-and-effacing (A/E) O157(+) bacteria were observed in 69% (38/55) of the STEC O157-inoculated calves. Rectum, ileocecal valve, and distal colon were sites most likely to contain A/E O157(+) bacteria. Fecal and intestinal levels of STEC O157 bacteria were significantly higher and A/E O157(+) bacteria were more common in DEX-treated calves than in nontreated calves inoculated with STEC O157. Fecal STEC O157 levels were significantly higher than Stx(-) O157, STEC O91, or control E. coli; only STEC O157 cells were recovered from tissues. Identifying the rectum, ileocecal valve, and distal colon as early STEC O157 colonization sites and finding that DEX treatment enhances the susceptibility of weaned calves to STEC O157 colonization will facilitate the identification and evaluation of interventions aimed at reducing STEC O157 infection in cattle.

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.

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.

Fifteen weaned calves (age 89-141 days) were treated with dexamethasone (0.25 mg/kg, IV) for 3 days before, the day of, and the day after inoculation with 10 colony-forming units of either Escherichia coli O157:H7 (strain 86-24, which produces Shiga toxin 2 and intimin; n = 13) or nonpathogenic E. coli (strain 123, which does not produce Shiga toxin or intimin; n = 2). All calves were necropsied 4 days after inoculation. Histologic lesions of attaching and effacing bacteria were observed in the large intestine (12/13) and in the gallbladder mucosa (5/13) of calves inoculated with E. coli 86-24. Cholecystitis was present in 12 of 13 calves that received E. coli 86-24. Inoculum bacteria were recovered from the distal colons or feces (13/13) and gallbladders (3/4) of calves inoculated with 86-24.

We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H(-) strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysin-encoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Inoculation of adult, female BALB/c mice with 2 X 10(5) bleach-treated Cryptosporidium sp. oocysts isolated from calf feces resulted in infection of the uterine mucosa in more than 50% of the animals. Cryptosporidium sp. completed the entire life cycle in the uterus, and infectious oocysts were passed into the vagina. Two methods of application were used to establish intrauterine infection. The inoculum was either injected into the uterus after abdominal surgery or intracervically instilled. Mice were susceptible at all phases of the sexual cycle, but the highest infection rates were obtained during estrus and diestrus. Parasites were demonstrated as early as 5 days postinfection. Phagocytic cells in the uterine lumen and in the vagina contained Cryptosporidium sp. Phagocytosis may be an important immune response and a mechanism of parasitic clearance. These results suggest that Cryptosporidium sp. is a potential pathogen of the reproductive tract.

Lymphoepithelium and cells specialized for uptake and transport of foreign matter are characteristic of antigen sampling organs, including lymphoglandular complexes (LGCs). Distribution, histologic structure, and epithelial ultrastructure of colonic lymphoglandular complexes were determined in 5- to 13-week-old pigs. LGCs averaged 1,231 in number per colon, displayed a characteristic distribution pattern, and were most evenly distributed in colons of older pigs. LGCs consisted of well-defined submucosal masses composed of lymphatic nodules and internodular lymphoid tissue penetrated by radially branching extensions of mucosal glands. Epithelial diverticula of each LGC entered the submucosa as a group through a circular collar derived from the muscularis mucosae. LGC epithelium contained goblet cells, cuboidal and columnar enterocytes, enteroendocrine cells, individual and clustered intraepithelial leukocytes, and cells morphologically compatible with follicle-associated epithelial cells/M cells. We regard the colonic LGC as a distinct mucosal lymphoid organ and suggest a significant role for it in local and systemic immune responses. The porcine colonic LGC may serve as a model for the human LGC.

Trypsin-digested paraffin tissue sections were used to demonstrate rotavirus particles in small intestinal epithelial cells of dirrheic neonatal calves, using direct fluorescent-antibody assay. The results were compared with other techniques to demonstrate rotavirus particles in tissue sections by immunofluorescence. Enzyme treatment of deparaffinized tissue sections gave excellent results with distinct specific fluorescence and minimal background. Prior staining of the tissue section with Mayer's hematoxylin made simultaneous observation of the same tissue section by light and ultraviolet microscopies possible.

The intestinal tissues from 11 pigs orally inoculated with Escherichia coli (E. coli, 0139:K12:H1) were examined by transmission electron microscopy. The colonization of E. coli along the small intestinal mucosa was found in seven principals without any major changes in the enterocytes from day 2 to day 7 after inoculation when the experiment was terminated. Lesions of vessels of the intestinal mucosa could be detected as early as two days after inoculation and persisted until the experiment was terminated. Lesions consisted of endothelial swelling and vacuolation, subendothelial fibrin deposition, perivascular edema, microthrombus formation, endothelial proliferation, and necrosis of the tunica media. The possible pathogenesis of the disease is discussed.

Cryptosporidium oocysts were found in the feces of 16 of 20 (80%) 17-day-old and 38 of 100 (38%) 24-day-old turkeys in a healthy commercial flock in central Iowa. Mean dimensions of 50 oocysts were 4.5 x 5.3 micron. In experimentally inoculated turkeys and chickens, the parasites frequently infected the ceca, colon, and cloaca, but rarely infected the small intestine or bursa of Fabricius. These data differ from published descriptions of Cryptosporidium baileyi in chickens and turkeys. The results indicate that the cryptosporidia of poultry are not a homogeneous group and demonstrate that Cryptosporidium infections can occur in apparently healthy poultry flocks.

Three isolates of bovine astrovirus, one from the United Kingdom and two from the United States, possessed common antigens by immunofluorescence and strain-specific antigens by neutralization and were designated as two, and probably three, distinct serotypes. The isolate US2, despite being a different serotype, possessed the same restrictive cell tropism and cytopathology as previously reported for isolate US1, of the M cells of the dome epithelium of the Peyer's patches. Serotyping of 16 field isolates indicated the presence of more undefined serotypes.

Three isolates of Breda virus of calves were compared morphologically and antigenically. The isolates demonstrated similar morphology and shared common antigens, as determined by enzyme-linked immunosorbent assay and immunoelectron microscopy. On the basis of results of the hemagglutination-inhibition test, enzyme-linked immunosorbent assay, and immunoelectron microscopy, the 3 isolates were further subdivided into 2 serotypes: serotype 1 (Breda virus 1) represented by the Iowa isolate 1; and serotype 2 (Breda virus 2), by the Ohio isolate and the Iowa isolate 2. The 3 isolates caused diarrhea in gnotobiotic calves.

Depressed lymphocyte blastogenesis in response to mitogen stimulation, depressed iodination of protein by neutrophils, and enhanced ingestion of Staphylococcus aureus by neutrophils were detected in a bull with chronic bovine viral diarrhea (BVD). Before developing chronic BVD, the bull was vaccinated with a killed cytopathic BVD virus. Neutralizing antibodies specific for the vaccine virus were detected in serum specimens obtained from the bull immediately before death. A noncytopathic BVD virus was isolated from the spleen after death. The immunologic and virologic findings in this bull supported reported research findings on the pathogenetic mechanisms involved in chronic BVD and mucosal disease.

In vitro motility and morphology of Cryptosporidium sporozoites were examined in the presence of various solutions. Crude preparations of the bile salt, taurocholic acid, maintained both motility and morphology in a dose-dependent manner. These effects appeared to be due to the taurocholic acid itself, and not simply due to pH variations, osmotic factors, or contaminants. Lysis of sporozoites was also observed and was found to be dependent on pH, with acidic conditions (pH less than 6.2) triggering the lysis.

Tracheal and conjunctival infections with Cryptosporidium were established in pigs by inoculation of oocysts into the trachea and onto the conjunctival sacs. The protozoa were found attached to epithelial cells by an electron-dense band and a folded, vacuolated feeder organelle. They were situated in a parasitophorous vacuole surrounded by a double-layered membrane covered by glycocalyx. Trophozoites, schizonts, merozoites, macrogametes and oocysts could be demonstrated. In addition to normal stages of the life-cycle, degenerate stages were found. The infections with Cryptosporidium were characterized by focal destruction and loss of epithelial cells. Numerous intraepithelial lymphocytes were associated with affected foci, as were infiltrations with lymphocytes, monocytes, and macrophages.

A bovine enteric virus antigenically related to the United Kingdom isolate of bovine astrovirus was isolated from diarrheic feces, also containing rotavirus, of a calf in Florida. The astrovirus infected cell cultures and the epithelial cells of domes in the ileum, and there was cross-immunofluorescence with antiserum to the United Kingdom astrovirus. Calves infected with astrovirus alone did not develop clinical disease, but when astrovirus was mixed with rotavirus or Breda virus 2, the calves developed severe diarrhea and more extensive astrovirus infection of the dome epithelium. The dome epithelial cells showed degeneration associated with astrovirus infection, and a few cells showed degeneration with Breda virus 2 infection. Virions with a 30-nm diameter were seen in astrovirus-infected dome cells, and Breda virus 2 virions were also observed either in separate cells or, on occasion, with both viruses in one cell.

In the present work, we attempted to characterize 4 field strains of Salmonella cholerae-suis var kunzendorf susceptible to antibody and complement (Ab-C), and 4 strains not susceptible to Ab-C, with respect to their virulence for mice and pigs. In vivo growth of an Ab-C-susceptible, mouse-virulent strain in the spleens of mice exceeded that of 2 Ab-C-susceptible, but mouse-avirulent strains by at least 10(4) organisms. Comparison of Ab-C susceptibility with virulence for pigs seemed to indicate that parallelism existed between Ab-C susceptibility and avirulence, except for strain 38, which was Ab-C susceptible yet virulent. There was agreement between virulence for mice and pigs, except for strains 10 and 61, which were avirulent for mice but relatively virulent for pigs. The 3 strain criteria under comparison (ie, Ab-C susceptibility, virulence for mice, and virulence for pigs) suggested only partial relatedness. Vaccination of pigs with the least virulent strain was found to protect them against challenge exposure with the most virulent strain, indicating that virulence factors may not be associated with immunogenicity.

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.

The pathogenicity of Cryptosporidium species was studied by inoculation of two gnotobiotic calves with cryptosporidial oocysts that had been decontaminated by treatment with peracetic acid. Two control calves were inoculated with similar material from which the oocysts had been removed by filtration. Oocyst-inoculated animals shed Cryptosporidium in their feces and developed depression, weakness, anorexia, and diarrhea. At necropsy five days after inoculation, endogeneous stages of Cryptosporidium were found in association with epithelial cells throughout the small and large intestines of these animals. The parasites were most numerous in the lower small intestine. Atrophic villi, disordered and degenerate villous epithelium, and hyperplastic crypt epithelium were associated with infection in the small intestine. Control animals remained normal. Extraneous agents were not detected in any of the calves. The results indicate that Cryptosporidium can destroy intestinal epithelial cells and cause diarrhea in monoinfected gnotobiotic calves.

Four gnotobiotic calves were inoculated intranasally with Breda virus within two hours after cesarean section. Three calves developed diarrhea between 48 and 60 hours after inoculation; one calf was killed at 36 hours. Consistent microscopic findings in the intestinal mucosae of calves with diarrhea were cytopathologic changes in enterocytes of the lower small intestine, large intestine, and dome epithelial cells and an acute inflammatory response with cellular infiltration and subtle changes in capillaries. Virions with average dimensions of 35 x 80 nm were seen in rounded cells that had severe cell swelling, dilatation of the cytocavitary network, and hydropic degeneration. Virions were most often in multiple, large autophagolysosomes. There was a good correlation in detection of virus in cells with electron microscopy and indirect immunofluorescence. No lesions were detectable in two control calves killed five days after inoculation.

The morphological aspects of Breda virus serotype 2 replication in intestinal cells of gnotobiotic calves were investigated by electron microscopy. Ultrastructural findings suggest a morphogenetic pathway involving cytoplasmic vesicles, the Golgi apparatus and the cell nucleus. Virus uptake probably occurs via a receptor-mediated endocytosis-like mechanism. Endocytotic vesicles then carry virus to an as yet undetermined site of uncoating. Masses of tubules having the same diameters as Breda virion cores are found in nuclei, suggesting a role for the cell nucleus in replication of nucleocapsids. Similar tubules, as well as complete virions, were found in the Golgi region, the apparent site of virus assembly. Virus-containing Golgi vesicles then presumably move to cell surfaces where they fuse with apical and baso-lateral cell membranes to release virions in a way that permits more than one viral replicative cycle to occur without damage to host cell integrity. Virions are elongated with rounded ends and measure 42 X 100.5 nm. The morphogenesis and replication of Breda virus most closely resembles that of Berne virus of the proposed family Toroviridae.

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.