Leukocyte numbers and intestinal mucosal morphometrics in horses with no clinical intestinal disease

Healthy horses and other animals have large numbers of resident leukocytes in the intestinal wall, but there is scant information regarding which and how many leukocytes are normally present in the equine intestinal wall. Our aim was to provide a reference range of leukocytes in the intestinal mucosal and submucosal propria of normal horses. We included in our study intestinal tissues from 22 Thoroughbred racehorses with no clinical intestinal disease, which had been euthanized because of catastrophic musculoskeletal injuries. Neutrophils, lymphocytes, eosinophils, macrophages, and plasma cells were counted in 5 random 17,600-µm² areas of villus lamina propria of the duodenum, jejunum, and ileum, and deep lamina propria of the duodenum, jejunum, ileum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon, and small colon. Other features investigated in the same intestinal segments included villus height and width (small intestine), presence of ciliated protozoa, Paneth cells number, subcryptal leukocyte layers (number of leukocyte layers between the bottom of the crypts and the muscularis mucosae), and submucosal leukocytes. Lymphocytes were the most numerous cells in all segments analyzed, followed by plasma cells, eosinophils, macrophages, and neutrophils. Eosinophil numbers were significantly higher in both lamina propria and submucosa of the large intestine than in the small intestine. The duodenum had shorter and thinner villi than either jejunum or ileum. The data provided from our study will be useful for diagnosticians examining inflammatory processes in the intestinal tract of horses.     

Eimeria organisms develop in the epithelial cells of equine small intestine

Histopathologic and immunohistochemical examinations were performed to determine the origin of host cells parasitized by Eimeria in the small intestines collected from five foals. Eimeria organisms at various stages (mainly microgametes and macrogametes) were frequently found in the cytoplasm of hypertrophied host cells in the lamina propria at the tips of villi of the jejunum and ileum. The cytoplasm of the host cell was immunohistochemically positive for cytokeratin AE1/AE3 and cytokeratin 13 and was negative for vimentin, desmin, alpha-smooth muscle actin, chromogranin A, neuron-specific enolase, and factor VIII. The host cells parasitized by Eimeria species had the immunostaining characteristics of epithelial cells but not of mesenchymal cells, endothelial cells of lacteals or capillaries, smooth muscle cells or neuroendocrine cells. These results suggest that the host cell of Eimeria species is possibly derived from intestinal epithelial cells and then displaced into the lamina propria of the small intestine.     

Intestinal lesions in specific-pathogen-free lambs associated with a cryptosporidium from calves with diarrhea

Small and large intestines of seven specific pathogen-free lambs infected with cryptosporidia from calves with diarrhea were examined by scanning and transmission electron microscopy and by light microscopy. The small intestine was infected in all the lambs, and the cecum and colon in three. Small intestinal alterations were severe villous atrophy and dilatation of the crypts of Lieberkühn. Epithelial cross-bridging between contiguous villi caused much villous fusion. Epithelial cells constituting the bridges were connected by desmosomal junctions, and were continuous with the epithelial coverings of the associated villi. The lamina propria was heavily infiltrated with neutrophil leukocytes. Infected crypts in cecum and colon were dilated and devoid of mucus-secreting cells, while the ridges between crypts were hypertrophied, and the lamina propria was infiltrated by neutrophils. Cell vegetations with adherent bacteria were present in the surface intestinal epithelium of two lambs infected for 11 and 14 days, respectively. No adherent bacteria were seen in any site in lambs killed up to six days post-inoculation.     

Scanning electron microscopic observations on the intestinal villi in growing White Leghorn and broiler chickens from 1 to 30 days of age

1. To obtain intestinal morphological data demonstrating the faster growth rate in broiler (BR) than in White Leghorn (WL) chickens, villi of the duodenum, jejunum and ileum were examined from 1 to 30 d of age by scanning electron microscopy. 2. At the first day after hatching, villi of each intestinal segment showed a finger-like shape in both breeds. Villi developed to a plate-like shape in the duodenum, a wave-like shape in the jejunum and a tongue-like shape in the ileum at 30 d of age via the common plate-like villi at 10 d of age. The fundamental villous shape and arrangement seem to be accomplished by 10 d of age; two types of obliquely elongated plate-like villi showed a zigzag arrangement, adjoining at an angle of 40 degrees to 60 degrees like an oblique T-shape. It is suggested that such a villous arrangement would be more effective for nutrient absorption by inducing a long zigzag flow of ingesta. 3. Compared with WL, even at the first day after hatching BR had many more developed epithelial cell protrusions over the whole apical surface of the duodenal villi. In WL the protrusions were not so apparent and located only in the central area of the villous tip. Furthermore, at 10 d of age BR showed more developed and larger villi, many wider microvilli at the apical portion of villi and more active extrusions of epithelial cells from the tip of the duodenal and jejunal villi. 4. These morphological characteristics of villi in early life in BR suggest a greater absorptive surface area and a more active intestinal function, permitting the faster growth rate of BR immediately after hatching.     

Scanning electron microscopy of intestine of gnotobiotic piglets infected with porcine rotavirus.

The development of intestinal lesions caused by the porcine rotavirus were studied in six day old gnotobiotic piglets by scanning electron microscopy. The onset of diarrhea followed an incubation period of 17 to 31 hr. The first detectable lesion was observed in the ileum at 12 hr postinfection, a few hours before the onset of diarrhea. At this time enterocytes appeared swollen and began to separate from each other. Seventeen hours after the onset of diarrhea, lesions were quite severe jejunum and ileum. Enterocytes were detaching from the lamina propria leaving denuded areas. Microvilli were sparse on the cell surfaces and there was marked villous atrophy. Regeneration of ileal mucosa was evident at 4.8 days after the onset of diarrhea. Nine days after recovery from diarrhea the intestinal villi had returned to near its normal structure but there remained some evidence of mucosal damage.     

肠艾美耳球虫配子生殖与病理变化

用单个肠艾美耳球虫Eimeria intestimalis Cheissin 1984卵囊感染无球虫兔,获得纯种进行研究。1.肠艾美耳球虫的配子生殖阶段寄生于空肠和回肠,此时宿主组织有较严重病变。12指肠、结肠和盲肠未见虫体,但在感染后264小时见盲肠的个别绒毛内有1～3个配子体,可能属偶然现象。2.感染后180小时发现极少数早期配子体,感染后192小时出现少量配子体寄生在空肠和回肠的绒毛和腺上皮细胞内,感染后216至264小时,绒毛上皮和腺上皮细胞内多为配子体、合子和卵囊所取代。感染后216小时出现极少量卵囊,264小时则见有大量卵囊。3.感染开始时(感染后61～73小时),回肠、空肠绒毛上皮正常;腺上皮细胞出现少量滋养体和裂殖体。96至192小时后,肠绒毛上皮和腺上皮受侵害程度渐趋严重,肠绒毛变矮,绒毛上皮及腺上皮细胞肿大变空,细胞核消失。许多腺泡塌陷。感染后216～264小时,肠绒毛受侵害最为严重,空肠和回肠绒毛萎缩或消失,变为一层矮柱或立方形上皮细胞或全无上皮细胞覆盖的绒毛。固有层均质红染,或颗粒状。肠腺塌陷,数量减少,大小不一。腺腔内见有配子体、合子或卵囊残留,部分腺泡上皮细胞再生,细胞核增生成堆。12指肠、盲肠和结肠正常。     

Microscopic alterations in jejunal epithelium of 3-week-old pigs induced by pig-specific, mouse-negative, heat-stable Escherichia coli enterotoxin

The objective of this study was to determine whether exposure of swine jejunum to crude culture filtrates containing Escherichia coli pig-specific, mouse-negative, heat-stable enterotoxin (STb) induces structural alterations in the jejunal mucosa of pigs. Two ligated intestinal loops in each of twelve 3-week-old pigs were exposed for 2 hours to sterile E coli culture filtrates from each of the following strains: 431 (STa-producing), 1261 (STa and STb-producing), and 1790 (STb-producing); recombinant strain HB101-pRAS-1 (STb-producing); the nontoxigenic K-12 variant HB101; or trypticase soy broth. Formalin-fixed sections from these loops were examined for sloughed cells around villi, and a lesion score was determined, indicating a change in villous epithelium from columnar to cuboidal or squamous cell types or to discontinuous epithelium. Villous lengths and crypt depths also were determined. For loops exposed to culture filtrates containing STa and STb or containing only STb, lesion scores and numbers of sloughed cells were greater (P less than 0.05) and villous length was shorter (P less than 0.01) than in loops not exposed to toxin. For loops exposed to culture filtrates containing STa, lesion scores, villus lengths, and numbers of sloughed cells were not different from those of loops not exposed to toxin. Therefore, exposure of swine jejunum to STb induced structural alterations in intestinal mucosa (ie, loss of villous absorptive cells and partial atrophy of villi) that were consistent with those causing compromised absorptive capacity.     

Immune development in jejunal mucosa after colonization with selected commensal gut bacteria: a study in germ-free pigs

The immunological structure of the porcine jejunal lamina propria in germ-free piglets was compared with that of their counterparts associated with two strains of commensal Escherichia coli, A0 34/86 serotype O83:K24:H31 and the O86 E. coli strain, up to 20 days post-colonization. In the antigen-presenting compartment, both dendritic cells (DC) and cells expressing CD163, probably macrophages were investigated. In addition we also assessed the number of CD2+/CD3+ (T) cells. In contrast to some previous reports, we show a total lack of both DC and T cells for germ-free animals in the diffuse lymphoid tissue of villi and crypts of the jejunum. Association with either strain of commensal E. coli had a profound effect on the immune structure and resulted in extensive recruitment of DC to the lamina propria and of T cells to epithelium and lamina propria. The data suggest that the earliest immigrant cells were monocytes, which soon acquired the phenotype of mucosal DC. T cells migrated in at a slightly slower rate. Nevertheless, the response could be extremely rapid: within 3 days of colonization with O83, the magnitude of this response was comparable to that observed 20 days post-colonization.     

Apoptosis of villous epithelial cells and follicle-associated epithelial cells in chicken cecum

The process of the disappearance of epithelial cells was examined in chicken cecal villi and follicle-associated epithelium (FAE). The apoptotic epithelial cells with intense DNA-fragmentation and their exfoliation were found in the villous tips. The epithelial cells with weak DNA-fragmentation were seen in the upper portion of the villi and their sparse exfoliations were also found there. Numerous epithelial cells in the intestinal lumen expressed the apoptotic features. A row of apoptotic epithelial cells with DNA-fragmentation was also found in the apical FAE, whereas no M cells exhibited any apoptotic signs. In all cecal regions, CD3+, CD8+, and TCR2+ lymphocytes were predominant in the epithelium at the upper portion of the villi and the FAE. CD4+ lymphocytes were mainly seen in the lamina propria. TCR1+ lymphocytes were not abundant in comparison with TCR2+ lymphocytes in the epithelium. TCR3+ T lymphocytes were rarely detected. These results suggest that the chicken cecal epithelial cells exfoliated into the lumen after the induction of the apoptosis, and that the induction may be involved with CD3+, CD8+, and TCR2+ lymphocytes. No death in M cells suggests that M cells may transform into microvillous epithelial cells.     

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.     

Studies on the pathogenesis of Salmonella heidelberg infection in weanling pigs

Experiments were conducted to define the pathogenic potential of Salmonella heidelberg in weanling pigs. Oral inoculation with S heidelberg resulted in severe catarrhal enterocolitis with accumulation of large amounts of fluid in the small intestine and colon. Salmonella heidelberg was demonstrated, with fluorescence microscopy and bacteriologic cultural techniques, to colonize the ileum, to invade ileal mucosal enterocytes, and to reach mesenteric lymph nodes and extraintestinal tissues by 8 hours. In 5 pigs, intestinal loops were surgically prepared and inoculated with S heidelberg (to determine its invasiveness). Microscopically, there were atrophy of villi, erosion of enterocytes, and neutrophilic infiltration in the lamina propria. Ultrastructurally, intracellular bacteria were demonstrated in villous and cryptal enterocytes, as well as in macrophages of the lamina propria. Bacteria were morphologically intact, occurred free and membrane-bound and caused no detectable cytotoxic effect to the cell.     

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.     

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.     

Transcytosis of F4 fimbriae by villous and dome epithelia in F4-receptor positive pigs supports importance of receptor-dependent endocytosis in oral immunization strategies

Very few antigens have been described that induce an intestinal immunity when given orally. Our laboratory demonstrated that oral administration of isolated F4 (K88) fimbriae of Escherichia coli to F4-receptor positive (F4R(+)) pigs induces protective mucosal immunity against challenge infection. However, presence of F4-receptors (F4R) on villous enterocytes is a prerequisite for inducing the immune response, as no F4-specific antibody-secreting cells (ASC) can be induced in F4R(-) pigs. In this study, the in vivo binding of isolated F4 fimbriae (F4) to the gut epithelium was examined in F4R(+) and F4R(-) pigs. It was further investigated whether binding of F4 to the F4R results in endocytosis in and translocation across the gut epithelium using microscopy. F4 did not adhere to the intestinal epithelium of F4R(-) pigs, whereas it strongly adhered to the villous epithelium and the follicle-associated epithelium (FAE) of the jejunum and ileum of F4R(+) pigs. Following binding to F4R, F4 was endocytosed by villous enterocytes, follicle-associated enterocytes and M cells. Transcytosis of F4 across the epithelium resulted in the appearance of F4 in the lamina propria and dome region of the jejunal and ileal PP. This is the first study showing transcytosis of fimbriae across the gut epithelium. This receptor-dependent transcytosis can explain the success of F4 fimbriae as oral immunogen for inducing protective immunity in F4R(+) pigs strengthening the importance of receptor-dependent endocytosis and translocation in oral vaccine strategies. Further identification of the receptor responsible for this transport is in progress.     

The Tissue Distribution of la- and IgM-Positive Cells in Adult and Newborn Miniature Pigs

Distribution of Ia- and IgM-positive cells was compared in tissues of adult and newborn miniature pigs, using indirect immunoperoxidase and immunofluorescent techniques with monoclonal antibodies. The different distribution patterns were found mainly in adult jejunum and kidney. Both IgM- and Ia-staining were ascertained in the lamina propria between the Lieberkühn crypts and in the lymphoid follicles of the jejunum. Moreover, IgM-positive cells were demonstrated in the crypts while Ia-reaction was seen in the lamina propria of the villi. In the kidney, IgM-staining was limited to the glomeruli. On the contrary, a distinct Ia-reaction was found mainly in intertubular structures. Small differences in IgM- and Ia-positive cell distribution were also noted in the mesenteric lymph nodes and in the spleen. Developmental changes of the adult distribution patterns (chiefly that of IgM-positive cells) were found in all tissues studied at 2 days of age.     

Lesions of the Gastrointestinal Tract of Pigs Infected with Transmissible Gastroenteritis

Gross, subgross and histological lesions were studied in 103 pigs infected with transmissible gastroenteritis virus and killed at daily intervals for 14 days. Twenty-three pigs served as controls. Thirty-six pigs were given colchicine four hours prior to being killed in order to determine the mitotic activity in the gastrointestinal tract. The gross lesions consisted of dehydration, excessive milk curd in the stomach, focal hemorrhage in the submucosa of the diverticulum ventriculi of the stomach, fundic and pyloric congestion in severly dehydrated animals and thinning of the small intestinal wall. The major subgross lesion was a marked shortening of the villi in the lower duodenum, jejunum and ileum within 24 hours after exposure to the virus. Regrowth of the villi became evident on about the sixth day after infection. Histological examination of the small intestine revealed that the villus-height/crypt-depth ratio in the jejunum was reduced from 7:1 in normal pigs to less than 1:1 in infected pigs. Villous atrophy was less severe in the proximal duodenum and ileum. Cells covering the atrophic villi were flatened or cuboidal and did not have well defined brush borders. Inflammatory changes in the gastrointestinal tract were minimal at all stages of infection. Goblet cell numbers increased slightly in the recovery stage of the disease and small numbers of mononuclear cells accumulated in the lamina propria during regrowth of the villi. The number of metaphase nuclei in the small intestinal crypts of infected pigs was greater than in normal pigs.     

Lymphocytic enteritis in a filly

A yearling Hanoverian filly had intermittent colic for 6 weeks, chylous peritoneal effusion, and a firm mass palpable per rectum. Exploratory laparotomy revealed mesenteric lymphadenopathy, adhesion of the mesenteric root to the duodenum and jejunum, distention of the mesenteric veins and lymphatic vessels, and increased jejunal venous pressure. Lesions in the duodenum, jejunum, and colon included infiltration of lymphocytes and plasma cells in the lamina propria.     

Supplementation of diets for lactating sows with zinc amino acid complex and gastric nutriment‐intubation of suckling pigs with zinc methionine on mineral status,intestinal morphology and bacterial translocation in lipopolysaccharide‐challenged weaned pigs

Sixty‐four pigs from 16 sows were used to evaluate addition of zinc amino acid complex (ZnAA) to lactating sows and gastric nutriment‐intubation of zinc methionine (ZnMet) to suckling pigs on mineral status, intestinal morphology and bacterial translocation after weaning. Sows were fed a barley‐based diet supplying 120 ppm zinc (Zn; control) or the control diet supplemented with 240 ppm Zn from ZnAA. At birth, day‐10 and day‐21 (weaning) of age, pigs from each litter were nutriment‐intubated with 5 ml of an electrolyte solution without or with 40 mg Zn from ZnMet. At weaning, 24 h prior to the collection of small and large intestinal lymph nodes and sections of the duodenum, jejunum and ileum, the pigs received an intramuscular injection of saline without or with 150 μg/kg body weight of Escherichia coli O26:B6 lipopolysaccharide (LPS). With the exception of a tendency (p = 0.09) for lower serum concentration of copper in pigs at weaning from ZnAA‐supplemented sows, there were no differences (p > 0.1) than for pigs from control‐fed sows for mineral status or intestinal morphology. Nutriment‐intubation of ZnMet increased serum (p = 0.001) and liver (p = 0.003) Zn concentrations, number of goblet cells per 250 μm length of jejunal villous epithelium (p = 0.001) and tended (p = 0.06) to enhance jejunum mucosa thickness. Interactive effects (p < 0.05) for higher jejunal villi height and villi:crypt ratio and increased ileal goblet cell counts were apparent for pigs from ZnAA‐supplemented sows that also received nutriment‐intubation of ZnMet. Challenge with LPS increased (p = 0.05) ileal villous width. Nutriment‐intubation of ZnMet decreased (p = 0.05) anaerobic bacteria colony forming unit counts in the large intestinal mesenteric lymph nodes. In conclusion, nutriment‐intubation of ZnMet increased serum and liver tissue concentrations of Zn and resulted in limited improvement to intestinal morphology of weaned pigs.     

Intestinal infection of neonatal dogs with canine coronavirus 1-71: studies by virologic, histologic, histochemical, and immunofluorescent techniques.

Enteritis induced in neonatal dogs by oral inoculation of coronavirus 1-71 was studied by virologic, histologic, histochemical, and immunofluorescent antibody methods. The enteritis, accompanied by diarrhea, developed in 4 to 7 days and was characterized by atrophy and fusion of intestinal villi and deepening of crypts, increase in cellularity of the lamina propria, flattening of epithelial cells, and discharge of goblet cells. Intra-epithelial enzyme activities generally were depressed. Specific immunofluorescent antibody reactions were present in villous epithelium of the duodenum starting on postinoculation day 2 and of the ileum on day 4. Epiehtlium of the colon showed no reaction. Virus was present in the feces for 6 to 9 days and was recovered from the small and large intestines from day 2 through day 10. The infection was not fatal and was self limiting; the diarrhea stopped between 1 and 2 weeks. The intestinal mucosa also started recovering by the end of week 1, proceeding caudally from the duodenum.