Ultrastructure and alkaline phosphatase activity of the dome epithelium of canine Peyer's patches

The dome epithelium of Peyer's patches from different parts of the intestine of four dogs was examined by scanning and transmission electron-microscopy and by alkaline phosphatase histochemistry on glycol-methacrylate embedded sections. M cells were scattered among more numerous enterocytes in duodenal and jejunal Peyer's patches, but constituted the major cellular component of dome epithelia of the ileal Peyer's patches. Alkaline phosphatase histochemistry demonstrated low enzyme activity in the brush border of M cells, as compared to enterocytes, in the duodenum and jejunum, allowing identification of M cells at the light microscopic level. Alkaline phosphatase activity was too low in ileal enterocytes to permit visualization of M cells. The presence of intrafollicular invaginations of dome epithelium is a consistent finding in duodenal Peyer's patches of the dog and these invaginations were characterized by few M cells, many intraepithelial lymphocytes and strong alkaline phosphatase activity.     

The intestinal habitat for organized lymphoid tissues in ruminants; comparative aspects of structure, function and development

Unlike the Peyer's patches of rats and mice, which are considered to be secondary lymphoid organs, the ileal Peyer's patch of sheep is thought to be responsible for the primary generation of B cells, like the bursa of Fabricius of birds. The ileal Peyer's patch of sheep shows prenatal maturation, antigen-independent lymphopoiesis, a rate of lymphocyte production larger than that of the thymus, and involution at a young age. Follicles contain few T cells and have an IgM+, relatively immature B lymphocyte population, as judged by B-cell differentiation markers. The follicle-associated epithelium of the ileal Peyer's patch is of a special type that sheds carbonic anhydrase-rich, 50-nanometer membrane-bounded particles (carbonic anhydrase-reactive particles; CAP) into the intercellular spaces. The CAP filter into the follicle centre and are taken up by lymphocytes. They represent the epithelial (bursa-like) element in an otherwise mesenchymal stroma of reticular cells embedding the follicle lymphocytes. Transepithelial transport of macromolecules, with the formation of multivesicular body-like cytoplasmic vacuoles, appears to be the basis for CAP formation. The jejunal Peyer's patches are devoid of CAP, persist in the adult animal, contain M cells with clusters of B cells in the follicle-associated epithelium, and have many CD4+ lymphocytes in the follicles and in the interfollicular areas. Aggregates of lymphoid follicles in the large intestine resemble the jejunal Peyer's patches with respect to their lymphocyte population and the ileal Peyer's patch with respect to their follicle-associated epithelium.     

Uptake of Mycobacterium avium subsp. paratuberculosis through the distal small intestinal mucosa in goats: an ultrastructural study

Various pathogens gain access to the intestinal wall via specialized cells, the M cells, found among the follicle-associated epithelial cells overlying the domes of the Peyer's patches. The present study was undertaken to examine the uptake of live Mycobacterium avium subsp. paratuberculosis in the distal small intestine of goat kids. Following laparotomy, distal small intestinal segments of five goats were ligated and injected with bacterial suspension. After 1 hour, the intestinal segments were excised and fixed for light and electron microscopic studies. M. a. paratuberculosis organisms were observed by transmission electron microscopy at locations in the intestinal wall, suggesting transcellular transportation through the M cells. The organisms were present both in the cytoplasm of the M cells and in the cytoplasm of intraepithelial leukocytes found in M-cell pockets. Intercellular bacteria between M cells were occasionally seen. Bacteria were not observed in association with the absorptive epithelium. This study indicates that in goat kids, M. a. paratuberculosis enters the intestinal wall primarily through the M cells in the follicle-associated epithelium of the Peyer's patches.     

Mycobacterium avium subsp. paratuberculosis enters the small intestinal mucosa of goat kids in areas with and without Peyer's patches as demonstrated with the everted sleeve method

The main lesions of paratuberculosis in ruminants are in the small intestine. Previous studies have shown that the bacterium enters the small intestine through M cells found in the follicle-associated epithelium lining the domes of the Peyer's patches. The everted sleeve method, devised for the in vitro study of intestinal absorption, was used in this study to investigate the uptake of Mycobacterium avium subsp. paratuberculosis in goat intestine. Everted small intestinal sleeves of goat kids, prepared from areas with and without Peyer's patches, were incubated for 60 min in 3H-labeled bacterial solution. The results of this study imply that the bacteria can enter the intestinal mucosa of the jejunum, both in areas with and without Peyer's patches. These findings indicate, therefore, that M. avium subsp. paratuberculosis bacteria not only enter through M cells but also through enterocytes.     

Apoptosis in normal lymphoid organs from healthy normal, conventional pigs at different ages detected by TUNEL and cleaved caspase-3 immunohistochemistry in paraffin-embedded tissues

The frequency and the distribution of apoptotic cells were investigated in formalin-fixed paraffin-embedded lymphoid tissues from healthy conventional pigs at four different ages (6 days, 2 months, 3.5 months and 5 months). Samples of tonsil, mesenteric lymph node, spleen, thymus and Peyer's patches were histologically processed and apoptosis evaluated with the TUNEL reaction and cleaved caspase-3 immunohistochemistry. In each technique, quantification of positive labelling was done for each particular lymphoid tissue area. The labelling pattern and distribution were similar for TUNEL and cleaved caspase-3. TUNEL stained mainly apoptotic bodies inside macrophages, but signal was also seen in free apoptotic bodies and in the nuclei of lymphocyte-like cells. The anti-cleaved caspase-3 antibody labelled mainly nuclei of lymphocyte-like cells. All tissues presented a similar distribution pattern of apoptosis, except for the 6-day-old group. In this group, a scattered distribution of positive cells was detected in tonsil, lymph node and spleen. In the tonsil and mesenteric lymph nodes from the older pigs, follicular areas presented higher amounts of positive cells than interfollicular areas. Moreover, the splenic white pulp showed more positive reaction than the red pulp, especially when they included germinal centres. In all groups, the follicular areas of ileal Peyer's patches presented more labelled cells than the dome and the lamina propria. In the thymus, the higher apoptotic rates were found in the cortex. In general, TUNEL yielded higher rates of positive cells than cleaved caspase-3 immunolabelling. A good correlation between the two techniques was found for thymus, tonsil and mesenteric lymph node, but not for Peyer's patches and spleen. This study describes a detailed histochemical characterization of apoptosis in pig lymphoid tissues using TUNEL and a cleaved caspase-3 immunolabelling at different ages. Moreover, our results indicate that TUNEL and cleaved caspase-3 techniques can be equivalent only when tissues have a high or low levels of apoptosis, since a considerable discrepancy was found in intermediate situations. Data from this study should be useful for future comparative studies under disease conditions.     

Distribution of T and B lymphocytes in jejunal and ileocaecal Peyer's patches of lambs

Jejunal (JPPs) and ileocaecal (IPP) Peyer's patches in lambs were studied by employing the indirect and the avidin-biotin-peroxidase methods, using antibodies to sheep IgM and thymocytes. Thymocyte sera absorbed with IPP germinal centre cells showed specificity for T cells in the thymus, lymph nodes and Peyer's patches. Whereas JPPs had large interfollicular T cell areas, IPP mainly contained B cells, although small triangular T cell areas were found at the apex of the follicles. Some T cells were also found in the dome and corona region of JPPs and IPP. While germinal centres of JPPs had about 40 per cent of IgM-positive cells, IPP germinal centres contained about 80 per cent of such cells. Negative or weak IgM-positive cells were seen in the peripheral zone of IPP germinal centres, contrasting with surface IgM-positive cells in the central zone, and indicating a centripetal migration of maturing lymphocytes.     

Ileal Peyer's patches in experimental infections of calves with rotaviruses and enterotoxigenic Escherichia coli: a light and electron microscopic and enzyme histochemical study

The effect of rotaviruses and enterotoxigenic Escherichia coli administered in various sequences to cesarean-derived, colostrum-deprived calves was studied using light and electron microscopy. The structure of the lymphoid tissue in the ileum, the number of mitoses in the crypts, number of intraepithelial lymphocytes, and enzyme histochemistry (alkaline phosphatase, acid phosphatase, succinic dehydrogenase, beta-galactosidase, and leucinaminopeptidase) of the ileal dome epithelium were evaluated. The area of lymphoid follicles in Peyer's patches of the ileum was investigated morphometrically. Monoinfections with either rotavirus or enterotoxigenic E. coli induced a significant increase in lymphoid follicle area, but did not affect dome epithelial cells. Dual infections did not consistently affect the follicle area, but the number of intraepithelial lymphocytes and the mitotic indices exceeded those of comparable monoinfections. Changes in activity of enzymes in the ileal dome epithelial area were minor.     

Follicle associated epithelium of the gut associated lymphoid tissue of cattle

The morphology of the gut-associated lymphoid tissue of the small and large intestine in three gnotobiotic calves was examined by scanning and transmission electron microscopy, and the distribution of specialized membranous cells present in the follicle associated epithelium was defined. Isolated follicles remaining in the ileum of a cow after involution of the continuous Peyer's patch were examined by scanning electron microscopy. The presence of membrane-bound particles, reported to be exclusively associated with the continuous Peyer's patch, was investigated in other gut-associated tissue of the small and large intestine of the calf. The presence of two types of follicle associated epithelium in the small intestine of the calf was confirmed, and the follicle associated epithelium of the large intestine proved to be a homogeneous population of specialized membranous cells, similar to that of the continuous Peyer's patch of the small intestine. In the discrete Peyer's patches, some specialized membranous cells were completely hidden by adjacent enterocytes and could only be identified by cytoplasmic extensions into the intestinal lumen. In the proximal part of the continuous Peyer's patch, a transitional zone was detected where the follicle associated epithelium of some doomed villi was composed of a homogeneous population of specialized membranous cells, while the epithelium covering other doomed villi consisted of a mixture of absorptive and specialized membranous cells, usually only found in the discrete Peyer's patches. Membrane-bound particles were observed associated with gut-associated lymphoid tissue in the small and large intestine.     

Investigation of bovine gut associated lymphoid tissue (GALT) using monoclonal antibodies against bovine lymphocytes

Gut associated lymphoid tissue of the small and large intestine of calves and cows has been compared morphologically and quantitatively using monoclonal antibodies to bovine lymphocytes. B cells were significantly decreased in the ileum of the cow compared to the calf. Significantly increased numbers of T cells were present in cell suspensions of all lymphoid areas of the cow compared to the calf. T lymphocyte subsets were quantified into cryostat sections of lymphoid tissues expressing BoT4, and BoT8 antigens demonstrated increased numbers in follicular and dome areas of the discrete Peyer's patches of the small and large intestine of the cow. BoT4+, BoT8+, and the non-BoT4/BoT8+ T cell subsets were increased in the mucosa of the cow as compared to the calf. Similarities in structure and lymphocyte composition of the discrete Peyer's patches of the small intestine, cecum and colon and isolated single follicles in the large intestine suggest similar functional properties.     

Effect of rotavirus and/or Escherichia coli infection on the aggregated lymphoid follicles in the small intestine of neonatal gnotobiotic calves

The effect of rotavirus and/or Escherichia coli infections on the follicle-associated epithelium (FAE or M cells) of the domes of the aggregated lymphoid follicles (ALF, or Peyer's patches) of gnotobiotic calves was evaluated by light, scanning electron, transmission electron, and immunofluorescence microscopies. Calf rotavirus (CRV) infection produced loss of FAE cell microvilli, and virions were observed in cytoplasmic vacuoles of FAE cells, as well as in intercellular spaces between FAE cells and lymphoid cells migrating through the dome epithelium. The CRV particles appeared to have entered the FAE cells by phagocytosis, with no subsequent cytoplasmic replication. Enterotoxigenic E coli (ETEC) induced more severe alterations including marked microvilli loss and ballooning in the FAE cells. There was no adhesion to, or colonization of FAE cells by ETEC, but bacteria were observed free or phagocytized within the dome and the germinal centers of the ALF. There were no ETEC observed in the cytoplasm of FAE cells. The presence of nonenterotoxigenic E coli (NETEC) in the intestine of calves had no effect on the intestinal FAE cells. The addition of NETEC to CRV infections did not enhance or modify in any way the response of FAE cells to the viral infection; however, the combination of CRV + ETEC produced severe necrosis of the FAE cells, and loss of dome epithelium of ALF.     

Characterization and Distribution of B Cells in the Lymphoid Organs of Goats

The distribution of B cells in the lymphoid organs of the goat was studied using a panel of 13 monoclonal antibodies (mAbs) developed against different markers for bovine B cells. Samples of mesenteric lymph nodes, jejunal and ileal Peyer's patches, duodenum, jejunum, ileum, colon, caecum and rectum were taken from four 7-month-old male Murciano-granadina goats using the avidin-biotin-peroxidase (ABC) method on frozen sections as described by Hsu et al. (1981). The mAbs against immunoglobulins (Ig) recognized a large number of cells, particularly in the light zones of the germinative centres of the lymphoid follicles, regardless of the isotype against which they were directed. However, the greatest numbers of B cells in the germinative centres and outer coronas of the lymphoid follicles of the lymph node, spleen and Peyer's patches were recognized by mAbs against the L lambda chain of Ig and against IgM. This was also the case in other locations where B cells were abundant, such as the medulla of the lymph node and the dome of the Peyer's patches. These mAbs recognized not only B lymphocytes but also plasma cells, showing an intracytoplasmatic reaction (numerous in the spleen red pulp and the intestinal lamina propria when mAbs were used against the L lambda chain of the Ig, scarce in the intestinal lamina propria when used against IgM and scarce in spleen red pulp and numerous in the intestinal lamina propria when mAbs against IgA were used). The mAbs BAQ44 A, GC65 A and GB25 A are of interest because, besides marking cells in the B areas where lymphocytes show surface Ig, they give a positive reaction in areas where there are Ig-cells (the dark zone of the germinative centre) and do not immunostain plasma cells. Thus, these mAbs recognize a surface marker which is not an Ig.     

Comparative studies on distribution and fine morphology of the intestinal Peyer's patches in Mongolian gerbils (Meriones unguiculatus) and mice.

The intestinal Peyer's patches (PP) were significantly different in distribution and fine morphology between Mongolian gerbils and mice. The PP of gerbils were evenly distributed from the duodenum to ileum, whereas the PP of mice were more densely distributed in the lower ileum than other parts of the intestine. In gerbils, each PP had a much greater number of lymphoid follicles than in mice, although the total number of PP was smaller. Electron microscopy revealed that the PP dome of gerbils was covered with two types of epithelial cells, one with shorter microvilli and the other with longer ones, whereas the epithelial cells of the murine PP dome was uniform-shaped with numerous medium-sized microvilli. Some dome absorptive cells of gerbils were morphologically similar to poorly differentiated crypt cells of mice, suggesting to be immature M cells.     

Morphology of intestinal colonization of Yersinia enterocolitica serovar O3 in mice

The present study was made to know the morphology of the initial invasion and lesions involved in the intestinal colonization of Yersinia enterocolitica serovar O3 in the epithelium of Peyer's patches of mice. Microfold (M) cells formed a specific structure like a pseudopodium and the bacteria were observed on the surface of the pseudopodium-like structure 4 hr after oral administration of serovar O3. The colonies of serovar O3 were observed in the epithelium and the lamina propria of the Peyer's patches dome region, and the bacteria grown in the Peyer's patches were in direct contact with the lumen without covered with the host tissue 24 hr after the administration.     

Morphological and functional comparisons of Peyer's patches in different parts of the swine small intestine

Fifteen conventional 8-week-old pigs were used to compare the morphology and function of Peyer's patches (PP) in different parts of the small intestine with special emphasis on the dome epithelium (DE). The comparisons were done by morphological observation through light and electron microscopy, and by the ability of the DE complex to phagocytize horseradish peroxidase (HRP). Dome epithelium of the PP in the jejunum was more superficially located in the mucosa in comparison with the ileum. The DE's of the ileum were much smaller, with an area of 3.7 micron2/DE, than that of the jejunum (18.4 micron2/DE). The number of DE areas/5 cm2 in the ileum was more than in the jejunum. However, the total surface area of DE/5 cm2 of PP, was larger in the jejunum (180.5 micron2) than in the ileum (55.6 micron2). Brown discoloration of diaminobenzidine-hydrogenperoxide (DAB+H2O2)-treated PP specimens, after HRP inoculation, intensified with post-inoculation time from 20 s to 5 min. The brown pigment first appeared on the surface of microvilli and infiltrated into the dome. No morphological differences were observed between the jejunum and the ileum in 1 micron thick Epon-embedded specimens. Intramucosally, brown pigment was almost always found in DE areas. The pigmented areas were more numerous in the jejunum but the color intensity showed no obvious difference. By transmission electron microscopy, the electron dense materials (which were interpreted as the products of HRP and DAB+H2O2) were found between the microvilli of membraneous (M) cells, in the intercellular spaces of the DE, and in a form similar to intracytoplasmic vesicles in the cytoplasm of M-cell and DE complex lymphocytes. Our results confirmed that DE of PP had much stronger phagocytic activity than did the ordinary villous epithelium. This evidence indicates that the DE complex of PP in the swine intestine is immunologically important.     

A comparison of histopathological changes in calves associated with K99- and K99+ strains of enterotoxigenic Escherichia coli.

Enterotoxigenic colibacillosis was experimentally produced in four colostrum-deprived calves given 10(10) Escherichia coli strain 210 (serotype 09+:K30+:K99-:F41-:H-) orally and the histopathological changes compared to those seen in colostrum-fed calves infected in an earlier study with strain B44 (serotype 09+:K30+:K99+:F41+:H-). Escherichia coli strain 210 caused diarrhea, atrophic villi with cuboidal epithelium, and focal accumulations of a few neutrophils in the dome villi above Peyer's patches but neither the clinical nor the histopathological changes were as pronounced as with strain B44. The extent and distribution of adherence to the mucosal surface differed between the two strains. Strain B44 adhered as a continuous layer over most of the absorptive epithelial surface of both the jejunum and ileum. Adherence of strain 210 was restricted to the ileum and the bacteria often adhered focally in "clumps" rather than as a continuous layer, especially on the distal half of the villous surface.     

Age-related changes in the anatomical characteristics of Peyer's patches in small intestine of Bactrian camels (Camelus bactrianus)

The distribution, size, and appearance of Peyer's patches vary according to species. In order to determine the anatomical characteristics of Peyer's patches in small intestine of Bactrian camel, and age-related changes in the number of Peyer's patches, 40 Bactrian camels of the following four age groups were studied: young (0.5–2 years), pubertal (3–5 years), middle-aged (6–16 years), and old (17–20 years). The exact number of Peyer's patches was recorded, and the appearance of Peyer's patches was described in detail. The results indicated that Peyer's patches of Bactrian camels not only have a particular anatomical location and distinct appearance but also change with age. They were distributed in the whole small intestine and there were four distinct types of Peyer's patches: nodular, faviform, cup-shaped, and cystic form Peyer's patches. However, the nodular and cystic form Peyer’s patches are specific to Bactrian camel, which have not been found in other animals including Dromedary camel. In addition, the distribution density of Peyer's patches in ileum was the maximum, then was jejunum and duodenum. Further statistical analysis showed that the number of Peyer's patches was altered with age. The number peaked in 5-year-old camels and declined subsequently with age. However, there was little change in the size of Peyer's patches in different age groups; no age-related macroscopic variations in the shape or size of the Peyer's patches were found. Results obtained from this study provide the basic information to further study on the gastrointestinal mucosal immunity of Bactrian camel.     

Distribution of the pores of epithelial basement membrane in the rat small intestine

The distribution and diameter of the pores of epithelial basement membrane in the intestinal villi and the lymph nodules of ileal Peyer's patches were investigated in the rat small intestine by scanning electron microscopy after the removal of the overlying epithelial cells with OsO(4) maceration. In the duodenum, jejunum and ileum, the pores were mainly distributed at the upper three fourths of the villi, but were scarce around the top of the villi. The diameter of some of the pores in the upper three fourths of the villi was larger than that of those in the lower portion. The protrusion of lymphocytes and the cytoplasmic processes of macrophages were also seen at the orifices of the pores. In ileal Peyer's patches, in contrast, pores were densely distributed in the lower one third of the follicle-associated epithelium (FAE) where M cells were mainly seen. Furthermore, these pores were larger than those found in the upper two thirds. Lymphocytes or cytoplasmic processes of macrophages were frequently seen in the lower one third of FAE. These results suggest that the pores at the basement membrane correspond to the passage of the immunocompetent cells which are in contact with M cells or villous columnar epithelial cells and that the abundance of pores is a sign of aggressive interaction between the particular epithelial cells and the immunocompetent cells at the upper three fourths of intestinal villi and the lower one third of FAE in the rat small intestine.     

Distribution of lymphocyte subsets in the small intestine lymphoid tissue of 1-month-old lambs

Distribution of lymphocyte subpopulations along the small intestine lymphoid tissue has been examined in 1-month-old lambs using flow cytometric and immunohistochemical techniques. Monoclonal antibodies against CD4, CD8, gamma delta, CD45R and B receptors have been employed in samples from continuous ileal Peyer's patch (IPP), discrete jejunal Peyer's patches (JPP), ileocaecal valve lymphoid tissue (ICVPP), mesenteric lymph node (MLN) and intra-epithelial (IEL) and lamina propria (LPL) lymphocytes. Histological studies were also done. Differences in the lymphocyte distribution have been observed between some of the regions examined, especially between IPP and JPP for most of the markers. A remarkable feature was the existence of morphological and lymphocyte distribution differences between ICVPP and IPP, locations that had been traditionally considered as similar. The antibody against CD45R receptor used in this study, that was supposed to mark B cells and some T cells, detected cell populations located in the dome of the follicles in all the samples, whereas the centre was negative. Lymphocytes positive to the B marker employed were located mainly in the centre, suggesting that both antibodies would mark B cells in different maturation status.     

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.     

Immunohistology of Peyer's patches in the dog.

Canine Peyer's patches were examined by light microscopy and immunohistochemistry for possible variations depending on the location within the small intestine and for similarities and dissimilarities to PPs from other species. The duodenal and jejunal PPs were characterized by relatively large domes and interfollicular areas. In contrast, the ileal PP had small domes and poorly developed interfollicular areas and very large follicles. T cells were found in the interfollicular area and corona and in lesser numbers in the dome and germinal centers. The ileal PP contained far fewer T cells than the proximal PPs. Domes of canine PPs contained some cytoplasmic IgA+ (cIgA+) and many cIgG+ cells. Peanut agglutinin (PNA) stained germinal center cells in a selective but not-uniform way and did not stain T cells.