Studies with enteroaggregative Escherichia coli in the gnotobiotic piglet gastroenteritis model.

Two strains of enteroaggregative Escherichia coli of human origin fed to gnotobiotic piglets caused diarrhea or death in the majority of them. Histological examination revealed moderate hyperemia of the distal small intestine and cecum, swelling of small intestinal villi, and layers of aggregated bacteria stacked together in a mucus gel-like matrix overlying intact epithelium. These findings confirm that enteroaggregative E. coli strains produce distinctive intestinal lesions different from those caused by other major categories of diarrheagenic E. coli.     

Effect of dietary regimen on rotavirus-Escherichia coli weanling diarrhea of piglets.

Previously, we induced weanling diarrhea in piglets by infecting them with rotavirus followed by hemolytic enteropathogenic Escherichia coli. We postulated that rotavirus, by damaging the epithelium of the small intestines, produced an enteroenvironment which favored the selection and growth of enteropathogenic E. coli. Furthermore, diet might affect the enteroenvironment and influence the course of the disease. To test this, newly weaned 3-week-old piglets were assigned to one of four dietary regimens and infected with rotavirus followed 24 h later with enteropathogenic E. coli. The course of the disease was followed by monitoring the severity of diarrhea and the fecal shedding of rotavirus and enteropathogenic E. coli in these dually infected piglets. The dietary regimen designed to tax the digestive and absorptive capacities of the piglets (high nutrient intake fed three times a day) produced the most prolonged diarrhea, colonization of the gut by hemolytic enteropathogenic E. coli, and persistent shedding of rotavirus (P less than 0.01). The same nutrient intake divided into 24 equal increments and fed hourly produced a less severe response (P less than 0.01). The least severe response was seen in piglets fed one-third the nutrient intake either hourly or three times a day (P less than 0.01). We conclude that dietary regimen plays an important role in rotavirus-E. coli-induced weanling diarrhea.     

Effects of Escherichia coli heat-stable enterotoxin STb on intestines of mice, rats, rabbits, and piglets

There are at least two classes of Escherichia coli heat-stable enterotoxins, STa and STb. Unlike STa, which is active in suckling mice and piglets, STb is inactive in suckling mice but active in piglets and older, weaned pigs. This study examined the activity of STb in several animals and its effect on intestinal histology and cyclic GMP levels in intestinal mucosal cells. STb did not cause fluid secretion in suckling mice up to 12 days old or in rat or rabbit intestinal-loop preparations. STb-induced fluid secretion in weaned-pig intestinal loops occurred by 30 min and became maximal by 3 to 6 h. STb did not disrupt intestinal histology and did not alter cyclic GMP levels in intestinal mucosal cells from piglet intestinal loops after 0.5- and 6-h incubations. Our studies support the concept that STb is a second heat-stable E. coli enterotoxin with properties and a mechanism of action unlike those of STa.     

Specific antibody and immunoglobulin responses after intestinal colonization of germ-free piglets with non-pathogenic Escherichia coli O86.

Colonization of the gut with components of commensal microflora profoundly affects the development of the immune system. The aim of the present study was to investigate mucosal and systemic B cell responses during the first few days after intestinal association of colostrum-deprived piglets reared in germ-free (GF) conditions with non-pathogenic Escherichia coli O86. Specific intestinal anti-E. coli antibodies (Ab), among which IgA Ab prevailed, were found 4 days after colonization (72% of standard) and their amount decreased 11 days later reaching 22% of standard. In contrast to mucosal Ab, specific serum Ab remained at the level of GF animals at day 4 (less than 10% of standard) and markedly increased 15 days after colonization (156% of standard). In addition to the occurrence of specific Ab, increased amounts of total immunoglobulins (Ig) of all isotypes were detected in sera and intestinal washings. Using the ELISPOT method an increased number of IgM, IgG and IgA-secreting lymphocytes were found in spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP) in colonized animals as compared to GF piglets. Contrary to cells from these lymphatic organs, B cells from thymus were not affected by E. coli stimulation. Our results show that at the onset of intestinal colonization, non-pathogenic E. coli specifically and polyclonally stimulate the mucosal and systemic humoral immunity, but relatively soon after stimulation, mucosal-specific responses in gut decreases, indicating the possible beginning of inhibition mechanisms (oral tolerance).     

The effect of intestinal colonization of germ-free pigs with Escherichia coli on calprotectin levels in plasma, intestinal and bronchoalveolar lavages

Calprotectin levels were measured by ELISA in plasma, terminal small bowel lavage and bronchoalveolar lavage from 8-day-old germ-free piglets or gnotobiotic piglets 24 h after colonization with one of the following Escherichia coli strains: non-pathogenic O86, probiotic Nissle 1917 or enteropathogenic O55. The concentration of calprotectin in plasma was about 30 ng/ml only in germ-free piglets and piglets associated with non-pathogenic E. coli. Piglets infected with O55 showed a significant increase of plasma calprotectin and the highest mean level of calprotectin in the bronchoalveolar lavage, which was coincident with septicaemia. However, in the lumen of the small intestine, E. coli Nissle 1917 alone elicited a significant increase of the calprotectin level which was confirmed by immunofluorescence and APAAP immunohistochemistry on cryostat sections through the small bowel. The relevance of this finding to the therapeutic effect of E. coli Nissle 1917 in inflammatory bowel disease is discussed.     

Chlorpromazine Reverses Diarrhea in Piglets Caused by Enterotoxigenic Escherichia coli

The effect of chlorpromazine (CPZ) on diarrhea caused by enterotoxigenic Escherichia coli was tested in piglets since CPZ has been shown to be a potent antagonist to enterotoxins in vitro in a cell system and in vivo in a mouse model. Experimental diarrhea was induced in three litters of newborn piglets which were infected by mouth with 2 x 10(9)E. coli bacteria, which produce heat-labile (LT) and heat-stable (ST) enterotoxins. Treatment with CPZ given intramuscularly 1 h after the onset of diarrhea reversed fluid secretion in small intestine as well as dehydration, as judged by clinical criteria. A dose of 5 mg of CPZ per kg of body weight completely normalized the intestinal-fluid content measured 4 h after diarrhea developed, whereas 1 to 2 mg of CPZ per kg of body weight was somewhat less effective but still caused significant reduction of fluid (P < 0.001). Studies with radioactive [(35)S]CPZ showed preferential and dose-dependent uptake of (35)S in the intestinal mucosa, the radioactivity being evenly distributed in the membranes of both crypt and villus cells. The enzyme adenylate cyclase, which probably mediates the cellular effects of LT, was shown to have two- to threefold higher activity in the infected than in the uninfected animals. This activation was reduced about 50% by the CPZ treatment (2 mg/kg of body weight). In a preliminary field trial the effect of CPZ was tested in a spontaneous outbreak of diarrhea in piglets due to enterotoxinogenic E. coli. The animals were treated either with oral electrolyte solution and standard antimicrobial agents only (controls) or with 1 mg of CPZ per kg of body weight intramuscularly in addition to this treatment. The mean duration of diarrhea in CPZ-treated animals was significantly shorter, 4.1 h (n = 23), than that in controls, 7.2 h (P < 0.05).     

益生菌抑制大肠埃希菌K1株黏附与侵袭的实验研究

目的评价三联活菌片中的益生菌对大肠埃希菌K1株在肠上皮黏附和侵袭的抑制作用。方法将大肠埃希菌K1株与Lovo细胞共孵育,采用黏附性抑制和竞争性排除方法,检测大肠埃希菌K1株黏附侵袭于Lovo细胞的数量,并采用流式细胞术分析益生菌对大肠埃希菌K1株诱导的细胞凋亡的抑制作用。将Sprague Dawley乳鼠随机分为实验组（益生菌和致病菌）与对照组（致病菌）。应用活菌计数法,对各组乳鼠肠道和血液中大肠埃希菌K1株进行定量检测,观察益生菌对大肠埃希菌K1株血行转移的拮抗作用。结果益生菌在体外能显著抑制大肠埃希菌K1株黏附侵袭于Lovo细胞（P〈0.01）,其抑制作用呈剂量依赖性;益生菌能显著降低大肠埃希菌K1株诱导的细胞凋亡（P〈0.01）;益生菌能显著抑制大肠埃希菌K1株的生长与血行转移,患菌血症的乳鼠数量显著降低（P〈0.01）。结论三联活菌片中益生菌能显著抑制大肠埃希菌K1株对肠上皮的黏附损伤和血行转移。     

Specific Antibody and Immunoglobulin Responses after Intestinal Colonization of Germ-Free Piglets with Non-Pathogenic Escherichia coli O86

Colonization of the gut with components of commensal microflora profoundly affects the development of the immune system. The aim of the present study was to investigate mucosal and systemic B cell responses during the first few days after intestinal association of colostrum-deprived piglets reared in germ-free (GF) conditions with non-pathogenic Escherichia coli O86. Specific intestinal anti-E. coli antibodies (Ab), among which IgA Ab prevailed, were found 4 days after colonization (72 % of standard) and their amount decreased 11 days later reaching 22 % of standard. In contrast to mucosal Ab, specific serum Ab remained at the level of GF animals at day 4 (less than 10 % of standard) and markedly increased 15 days after colonization (156 % of standard). In addition to the occurrence of specific Ab, increased amounts of total immunoglobulins (Ig) of all isotypes were detected in sera and intestinal washings. Using the ELISPOT method an increased number of IgM, IgG and IgA-secreting lymphocytes were found in spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP) in colonized animals as compared to GF piglets. Contrary to cells from these lymphatic organs, B cells from thymus were not affected by E. coli stimulation. Our results show that at the onset of intestinal colonization, non-pathogenic E. coli specifically and polyclonally stimulate the mucosal and systemic humoral immunity, but relatively soon after stimulation, mucosal-specific responses in gut decreases, indicating the possible beginning of inhibition mechanisms (oral tolerance).     

Attaching and effacing adherence of Vero cytotoxin-producing Escherichia coli to rabbit intestinal epithelium in vivo.

Vero cytotoxin-producing Escherichia coli (VTEC) strains have been associated with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome in humans. Since adherence of enteric pathogens to epithelial surfaces is often a prerequisite for the subsequent delivery of bacterial enterotoxins and mucosal invasion, we evaluated intestinal adherence by 18 VTEC strains, which were of human origin and belonged to 10 distinct serotypes, 7 days after enteral challenge of rabbits. A total of 23 postweaning rabbits (body weight, 1 kg) were each fed 2 X 10(8) VTEC, and 5 rabbits were challenged with an equal number of fecal commensal E. coli strains as controls. Each rabbit was monitored daily for the development of diarrhea. At 7 days after infection the proximal jejunum, distal ileum, cecum, and proximal colon were removed from each rabbit and examined for the presence of adherent organisms under light microscopy, after Giemsa staining of Formalin-fixed secretions, and by transmission electron microscopy. Nonbloody diarrhea developed in 16 of 23 VTEC-infected rabbits in contrast to 0 of 5 infected with commensal E. coli strains (P less than 0.02). Organisms were adherent to surface epithelial cells in the ceca (20 of 23 rabbits), proximal colons (9 of 23), and distal ilea (6 of 23) of VTEC-infected rabbits. Intimate attaching and effacing binding of bacteria to intestinal epithelial cells, in regions where the normal microvillous membrane architecture had been disrupted, was observed under electron microscopy for VTEC of multiple serotypes. In contrast, no organisms were adherent to the jejuni. Adherence of organisms was not seen in any portion of the intestines of rabbits that were challenged with commensals. These findings indicate that multiple serotypes of VTEC demonstrate intimate attaching and effacing binding to rabbit enterocytes and colonocytes in vivo. In addition to bacterial binding in the ceca and colons, VTEC adhere to enterocytes in the distal small intestines of per orally infected postweaning rabbits.     

Pathological changes in broiler chickens fed ochratoxin A and inoculated with Escherichia coli.

A study was conducted to evaluate the effects of ochratoxin A (OA) on Escherichia coli-challenged broiler chickens. One hundred and eighty-four one-day-old broiler chicks were divided into two groups of 92 chicks each, with one group fed a control mash diet and the other fed a mash diet containing 2 parts/10(6) OA. On day 14, each group was further subdivided into two groups with one group inoculated with E. coli O78 (1 x 10(7) colony-forming units/0.5 ml) by the intraperitoneal route, whereas the other group was not inoculated with E. coli. Four birds from each group were sacrificed at 1, 2, 3, 5, 7, 10, 14 and 21 days post-inoculation to record pathological changes in the liver, kidneys, heart, lungs, bursa, spleen and thymus. E. coli infection induced perihepatitis and pericarditis in the liver and heart, respectively, in chickens infected with E. coli alone or in OA-fed birds from 1 day post-infection (DPI) onwards. At 1 DPI, a thin fibrin layer covered the liver and heart; however, at subsequent days, the layer became thicker. E. coli infection did not produce appreciable changes in the kidneys, bursa or thymus. However, there was congestion of the lungs along with mononuclear cell infiltration. Ochratoxin feeding induced changes from 10 DPI onwards in chicks fed OA alone and those infected with E. coli. The changes in kidneys included swollen proximal convoluted tubules, degeneration of tubular epithelium and interstitial nephritis. Degenerative changes and mononuclear cell infiltration were recorded in the liver. There was atrophy of the lymphoid organs along with depletion of lymphocytes. Gross and histopathological changes were more severe in chickens fed OA and inoculated with E. coli than the chickens fed OA alone or those infected with E. coli, indicating combined action of these two.     

Prenatal and postnatal development of the large intestine in the insectivore Suncus murinus, the laboratory shrew

Development of the large intestine in the insectivore Suncus murinus (the laboratory shrew) was investigated from day 21 to 30 of gestation and from birth to 20 days of age. Two days before birth, the stratified epithelium in the large intestine changed into a single layer. Although neither villi nor villus-like structures were ever present, fissures, corresponding to openings of the crypts, appeared on the mucosal surface before birth. These increased in number as well as in width and depth, connected with each other, and gave the mucosal surface a ridge-like appearance by 20 days of age. An elevation containing submucosae appeared shortly after birth and formed a large circular fold during the neonatal period. Goblet cells were the predominant epithelial cell type. Individual epithelial cells were mature-looking a few days before birth; goblet cells contained numerous mucous globules and absorptive cells possessed well-developed organelles. However, although goblet cells increased in number and exhibited active mucous-releasing forms after birth, absorptive cells never showed morphologic evidence of active endocytosis, such as apical endocytotic complexes and large supranuclear vacuoles. Each epithelial cell was similar in ultrastructure to that of the adult shortly after birth.     

Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli O157:H7 in gnotobiotic piglets.

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has two putative virulence factors: (i) a fimbrial adhesin, specified by a 60-megadalton (MDa) plasmid, and (ii) bacteriophage-specified cytotoxin(s), known as Shiga-like toxin (SLT) or verotoxin. The contribution of these factors to the pathogenesis of EHEC-induced disease in gnotobiotic piglets was examined. The bacterial strains included the following: two EHEC strains and their corresponding plasmid-cured derivatives; another EHEC isolate and its derivative which had spontaneously lost the ability to produce SLT; one E. coli K-12 transconjugatant containing a 60-MDa plasmid from an EHEC strain; two K-12 strains into which an SLT-producing phage had been transduced (one of these strains also carried a 60-MDa EHEC-derived plasmid); and the parent K-12 strain. Each strain was fed to four piglets, which were observed for diarrhea and examined for development of characteristic mucosal lesions 3 or 5 days after inoculation. All 24 piglets inoculated with the three EHEC strains and their respective derivatives (two plasmid cured and one SLT negative) showed the typical mucosal lesions of bacterial attachment: effacement of microvillous border and cell membrane dissolution culminating in destruction of surface and glandular epithelium in the cecum and colon. No such lesions were observed in 12 piglets inoculated with three strains of E. coli K-12, including the strain which carried both the 60-MDa plasmid and a phage which specified production of SLT. Moderate to severe diarrhea was observed in 16 piglets inoculated with two EHEC strains and their derivatives (one plasmid cured and one SLT negative). The third EHEC strain and its plasmid-cured derivative produced fewer typical mucosal lesions and no diarrhea. The reason for the reduced virulence of this strain was not clear. These results demonstrate that neither the 60-MDa plasmid nor the capacity to produce SLT is essential for expression of virulence by E. coli O157:H7 in gnotobiotic piglets.     

Passive protective effect of chicken egg yolk immunoglobulins against experimental enterotoxigenic Escherichia coli infection in neonatal piglets.

Passive protection of neonatal piglets against fatal enteric colibacillosis was achieved with powder preparations of specific antibodies against K88, K99, and 987P fimbrial adhesins of enterotoxigenic Escherichia coli. The antibody powders were obtained by spray drying the water-soluble protein fraction of egg yolks from immunized hens after the lipid components were precipitated with an aqueous dispersion of acrylic resins (Eudragit L30D-55; Rohm pharma). The anti-K88, -K99, and -987P antibody preparations reacted specifically against the corresponding fimbrial antigens in an enzyme-linked immunosorbent assay. The orally administered antibodies protected in a dose-dependent fashion against infection with each of the three homologous strains of E. coli in passive immunization trials with a colostrum-deprived piglet model of enterotoxigenic E. coli diarrhea. Scanning electron microscopy revealed adherence of enterotoxigenic E. coli in intestinal epithelial surfaces of control piglets, whereas in treated piglets treated with high-titer antibodies, a resistance to bacterial adhesion was observed. An enzyme immunoassay with avidin-biotin complex demonstrated specific local antibody activity in target areas of the small intestines. In vitro, E. coli K88+, K99+, and 987P+ strains adhered equally to porcine duodenal and ileal epithelial cells but failed to do so in the presence of homologous anti-fimbrial antibodies. Absorption of egg yolk antibodies with fimbrial immunosorbent removed the anti-fimbrial antibody fraction and reduced significantly the protective nature of the antibody preparation in a passive immunization experiment, suggesting that anti-fimbrial antibodies were the active components.     

The gnotobiotic piglet as a model for the pathogenesis of Campylobacter jejuni infection

The pathogenesis of enteric changes was studied in gnotobiotic piglets which, after hysterectomy had been infected orally with Campylobacter jejuni on the first day of their life. The involvement of the entire large intestine became clinically manifest by scouring on days post infection (DPI) 4 to DPI 5, and pathomorphologically, by simultaneous inflammation and severe edema of the intestinal wall. Histology and SEM revealed inflammatory edema with abundant neutrophils, microulcerations, focal propagation and activation of goblet cells, and a presence of mucin-positive material within the intestinal lumen. TEM examination revealed disconnected interdigitating folds and wide dilated intercellular spaces between enterocytes. The endothelial cells of small blood vessels in the lamina propria showed hypertrophy with increase in the thickness of their basal lamina. Ultrastructural lesions of the large intestinal microcirculation also support the hypothesis that disturbances in the vascular system are responsible for edema in the cecum and colon. Gnotobiotic piglets may be used as a suitable animal model to study colitis induced by C. jejuni.     

Prenatal and postnatal development of the small intestine in the insectivore Suncus murinus

The development of the small intestine in the insectivore Suncus murinus was noted during the period from 21 days' gestation to 20 clays after birth. At 21 days of gestation, the proximal small intestine exhibited the beginning of villus formation, whereas the distal small intestine preserved the stratified epithelium. Stratified epithelium in the distal small intestine changed into a single layer by 24 days' gestation. At 26 day's gestation, each epithelial cell was immature; but by 28 days mature-looking epithelial colls were found. The shape of the villi changed from cuboid to columnar during the same period. The connective-tissue cores of the villi began to develop at 7 days after birth in the proximal small intestine and at 15 days after birth in the distal small intestine. Crypts appeared at 15 days after birth. Endocytosis of epithelial cells took place at 28 days of gestation. In the proximal small intestine, supranuclear vesicle clusters were observed first at birth; they began to decrease both in number and size at 10 days' gestation and then disappeared completely by 20 days after birth. In the distal small intestine, large supra-nuclear vacuoles were observed first at 28 days of gestation. Although these vacuoles invariably were found up to 15 days after birth, they also disappeared completely by 20 days. Epithelial cells showed a structure similar to those of the adult after weaning.     

Nature and distribution of mucosal lesions associated with enteropathogenic and enterohemorrhagic Escherichia coli in piglets and the role of plasmid-mediated factors.

Bacterial attachment-effacement (att-eff) is emerging as an important virulence characteristic common to both enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). The contribution of the plasmid-encoded EPEC adherence factor to the production of mucosal lesions and diarrhea was investigated in gnotobiotic piglets. Bacterial att-aff in the intestinal mucosa of piglets infected with plasmid-cured EPEC strain E2348/69 (O127) was indistinguishable from that in piglets infected with the parent strain, but the distribution of lesions was different; it occurred in the small intestines of 6 of 7 piglets infected with the parent strain compared with only 2 of 11 (P = 0.006) infected with the plasmid-cured strain. Plasmid-encoded factors in EPEC and EHEC strains did not appear to contribute to bacterial competition with normal gut microflora. Of 13 strains belonging to five EPEC serogroups, O55, O142, O26, O119, and O111, 3 fulfilled the criteria for EHEC (2 O26 and 1 O111). There were three distinct patterns of bacterial association with the intestinal mucosa of infected piglets. (i) EHEC strains caused bacterial att-eff associated with extensive destruction of surface and glandular epithelia in the large intestines with little or no inflammatory response. (ii) Some EPEC strains caused severe diarrhea which correlated with the extent of bacterial att-eff in the proximal small intestine, disruption of the epithelial cell membrane, and inflammation. It is suggested that, with respect to virulent strains, this degree of involvement determines the clinical outcome. Mildly pathogenic strains (O127 and O119), in which bacterial att-eff was restricted to the distal halves of the small and large intestines, caused little or no diarrhea. In such strains, nonimmune host factors (smaller, poorly feeding, and lethargic piglets) tended to play a determining role with regard to the degree of involvement of the small intestine and hence the clinical outcome. (iii) One strain (O55) caused illness and mucosal damage which could not be accounted for by the sparse bacterial att-eff observed in the gut. Instead, bacteria penetrated into and proliferated in the lamina propria, undermining the villous tips in the small intestine. Bacterial att-eff was the most important virulence factor in most of the strains examined, but plasmid-mediated factors facilitated bacterial adhesion in the small intestine, which may explain the reduced pathogenicity of the plasmid-cured variant of strain E2348/69 for human volunteers.     

Attaching and effacing enteropathogenic Escherichia coli O18ab invades epithelial cells and causes persistent diarrhea.

A case of persistent diarrhea following Escherichia coli O18ab gastroenteritis is reported. Electron microscopy of a biopsy of the small intestine showed effacement of the brush border, attachment of bacteria to the epithelial cells with pedestal formation, and bacteria within the enterocytes. The bacterial isolate was an enteropathogenic E. coli isolate which did not contain the adherence factor (EAF) but possessed the attaching-effacing eae gene, was able to invade HeLa cells in a gentamicin invasion assay, and also invaded rabbit intestinal cells. Results suggest that E. coli organisms of the O18ab serotype may cause diarrhea by an as yet unknown pathogenic mechanism, involving attaching to and effacing of enterocytes followed by invasion of the epithelial cells.     

Supplementation of Vitamin C to Weaner Diets Increases IgM Concentration and Improves the Biological Activity of Vitamin E in Alveolar Macrophages

Vitamins E and C have been found to increase the cellular and humeral immunity of pigs. Vitamin E deficiency has also been found to predispose pigs to different diseases, E. coli infection is one among them. After weaning, the vitamin E status of pigs often decreases to a critical low level. In this experiment, we studied whether vitamin C supplementation would be a possible feeding strategy to optimize the immune status of weaners. The interaction between vitamin E and C is interesting due to the reported sparing action on vitamin E or synergism between these to vitamins. Piglets were weaned at day 28 of age from sows fed increasing dietary vitamin E during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg STAY-C per kg. Blood sampling was obtained weekly from day 28 and until day 49 of age. On the same days, one piglet per dietary treatment was killed and alveolar macrophages (AM) were harvested. Vitamin C supplementation increased the concentration of IgM in serum of piglets throughout the weaning period. Although the vitamin E concentration in AM decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin E. However, vitamin C supplementation tended to increase the total AM concentration of vitamin E after weaning and increased the proportion of the biologically most active isomer of vitamin E [RRR-(α-tocopherol)] in the AM. The eicosanoid synthesis by AM was not influenced by the vitamin C supplementation, but the synthesis of leukotriene B4 was decreased 2 weeks after weaning compared to other days of AM harvesting. In conclusion, dietary vitamin C supplementation improved the immune responses of piglets after weaning.     

Quantitative assessment of the ability of Escherichia coli to invade cultured animal cells.

Assays to quantify bacterial invasion of epithelial cells generally fail to take account of the ability of the bacteria to adhere to the cells prior to invasion. We have developed a modified invasion assay to allow for this factor. We then used the assay to investigate diarrhoeagenic strains of Escherichia coli with differing ability to adhere to and invade HEp-2 epithelial cells. The results showed that enteroinvasive strains of E. coli were the most invasive variety, followed in order by enteropathogenic E. coli and enterotoxigenic E. coli. These findings correspond to what is known of the ability of the bacteria to invade the intestinal tract in vivo. The results also indicated that adhesins of diarrhoeagenic E. coli play no direct role in invasion, although they may facilitate invasion indirectly by promoting initial contact between bacteria and animal cells.     

Enhancement of Susceptibility to Shiga Toxin-Producing Escherichia coli O157:H7 by Protein Calorie Malnutrition in Mice

Infection with Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli is increasing among children. In this study, 5-week-old C57BL/6 mice with protein calorie malnutrition (PCM) that had been fed a 5% protein diet for 2 weeks since ablactation were inoculated intragastrically with 2 × 10⁶ CFU of Stx-producing E. coli O157:H7. More than 75% of infected mice with PCM died by 10 days postinfection. Infected mice with PCM developed neurologic symptoms 5 days after infection, while well-nourished control mice receiving a 25% protein diet did not. In the intestinal tracts of infected mice with PCM, inoculated E. coli O157:H7 multiplied between days 2 and 4 of infection, with a peak of growth at day 4. Although the pathogens were not culturable from the stool after day 7, O157 lipopolysaccharide was detectable in the stool by enzyme-linked immunosorbent assay even after day 8. Stx was detectable in the stool after day 2 of infection and increased in proportion to the growth of inoculated organisms. The maximal production of Stx occurred at 4 days postchallenge, and Stx was detectable in the blood on days 3 to 5. In contrast, well-nourished control mice survived the infection, and all of them remained well even after 3 weeks of infection. In these control mice, inoculated E. coli O157:H7 disappeared from the stool before day 3. Stx was not detectable in the stool and blood of infected control mice at any time from day 1 through day 8. Histologically, cerebral hemorrhages seemed to be the cause of acute death of infected mice with PCM. Immunocytochemical staining demonstrated the positive immunoreaction to Stx at the alveus and stratum pyramidale of the hippocampus and in renal tubules of infected malnourished mice. Such immunoreactions were not found in tissues from infected control mice. Histological study of the intestinal epithelium before infection showed that PCM severely affected the development of intestinal epithelia. These findings strongly indicate that PCM-induced nondevelopment of intestinal physical barrier is one of the predisposing factors for infection with Stx-producing E. coli O157:H7 in mice and suggest that our mouse model may explain the high incidence of infection with Stx-producing E. coli O157:H7 in the children whose intestinal epithelia have not yet completely developed.