Protective efficacy of a genetic subunit bacterin against edema disease of swine in mice.

The exotoxin SLT-IIeB from the Escherichia coli Ee strain was expressed in E. coli, and the recombinant protein was purified, mixed with the Ee strain, then emulsified with oil-emulsion adjuvants to obtain a mixed subunit bacterin. Groups of Kunming mice were immunized at weeks 0 and 2, and challenged intraperitoneally with the Ee strain at week 4. Antibodies were detected by ELISA and an agglutination test. After the second immunization, the antibody level increased and the rate of immune protection against the Ee strain was 70 and 91.7% in the subunit bacterin and bacterin groups, respectively. Therefore, the mixed subunit bacterin provided good protection against the homologous Ee strain, which provides a basis for further research, into high-efficacy vaccines against porcine edema disease.

Experimental infection of enterotoxemic Escherichia coli associated with porcine edema disease and its pathologic characteristics in the intestine.

Edema disease (ED) has become frequent in Japan, but no effective method for experimental infection has been developed. We report here the use of a capsule that resistant against gastric digestion to induce the ED in piglets. Four 21-day-old piglets were used. Shiga toxin 2e-producing Escherichia coli (STEC) cell pellet was encapsulated and administered orally. Two pigs received 1.0x10(10) CFU for two days, and the others received 3.9x10(8) CFU for three days. The high-dose group caused the typical clinical ED signs (palpebral edema or neurologic impairment). Eosinophil infiltration, swollen lymphoid follicles, and edema were observed in the ileum. The kidney had the thrombus in the glomerulus.

The effect of organic acids on the control of post-weaning oedema disease of piglets.

Oedema disease usually occurs after weaning and is due to infection with Enterotoxaemic Escherichia coli strains. A total of 240 weaned piglets were used in five groups during a 28-day period. One group (a negative control) was offered feed free of antimicrobials ad libitum, three groups were offered the same diet ad libitum supplemented with either 1.6 per cent lactic acid, 1.5 per cent citric acid or 50 p.p.m. of enrotloxacin (ENR/Baytril I.E.R. 2 5 per cent, Bayer), respectively. Finally, one group was offered the same diet but the amount offered was restricted during the first 12 days post-weaning. Groups receiving acid or ENR additions to the diet had lower mortality than the negative control group (P<0.05). The three groups on treated feed also showed significantly better growth performance and food conversion ratio than the control group (P<0.05). Both organic acids and medication with 50 p.p.m. of ENR for a 10-day period are useful in controlling and/or preventing post-weaning oedema disease.

Genetically modified Shiga toxin 2e (Stx2e) producing Escherichia coli is a vaccine candidate for porcine edema disease.

Porcine edema disease (ED) is an enterotoxaemia in pigs after weaning, caused by Shiga toxin 2e (Stx2e) producing Escherichia coli. Recently in Japan, outbreaks of ED are re-emerging in pig production. In this study we constructed a mutant that retained immunogenicity but lost Vero cell cytotoxicity, which produced genetically modified toxin (Stx2e*) by replacing glutamate with glutamine at position 167 and arginine with leucine at position 170 of the A subunit. The stx(2e)* gene was replaced with the stx(2e)gene of the wild type virulent strain by homologous recombination. As the parent wild strain was pathogenic to pigs but the mutant was not, the mutant named as YT106 was given to the pigs to examine its protective immunity against ED. All 20 pigs vaccinated with YT106 survived, but only eight of the 20 non-vaccinated pigs survived after the challenge with a wild strain. Also, the eight pigs that survived had decreased rates of gain relative to those of the controls. Blood IgG and intestinal IgA titres increased 3.3 and 1.6 times more than the control, respectively, showing that YT106 might be a good candidate of a live attenuated vaccine strain to protect against ED.

Shiga toxin-producing Escherichia coli infection: temporal and quantitative relationships among colonization, toxin production, and systemic disease.

Edema disease, a naturally occurring disease of swine caused by Shiga toxin-producing Escherichia coli (STEC), was used as a model for the sequence of events that occur in the pathogenesis of STEC infection. The mean time from production of levels of Shiga toxin 2e (Stx2e) detectable in the feces (day 1) to the onset of clinical disease (neurologic disturbances or death) was 5 days (range, 3-9). Bacterial colonization and titers of Stx2e in the ileum peaked at 4 days after inoculation in pigs without signs of clinical disease and at 6 days after inoculation in clinically affected pigs. Animals with the greatest risk of progressing to clinical disease tended to have the highest fecal toxin titers (>/=1:4096). Stx2e was detected in the red cell fraction from blood of some pigs showing clinical signs of edema disease but was not detected in the serum or cerebrospinal fluid.

Ultrastructure and DNA fragmentation analysis of arterioles in swine infected with Shiga toxin-producing Escherichia coli.

Shiga toxins (Stx) produced by E. coli are potent cytotoxins that affect the vascular system. In humans, systemic toxemia causes renal glomerular damage manifested as hemolytic uremic syndrome. In swine, Stx-producing E. coli (STEC) cause edema disease that is characterized microscopically by segmental arteriolar smooth muscle cell (SMC) lesions. Our objectives were to characterize ultrastructurally and by TUNEL the type of death (apoptosis or necrosis) that occurs in SMCs during edema disease. Increased DNA fragmentation consistent with apoptosis was detected by TUNEL in arterioles of challenged pigs 14-15 days post inoculation. Ultrastructurally 3 grades of SMC lesions were distinguished: 1) Partial loss of SMCs, intercellular space filled with granular cellular debris admixed with membrane bound vacuoles; 2) Complete loss of SMCs; only granular cellular debris and clear vacuoles remained within basement membrane; 3) Inflammation of media; SMCs replaced by a rim of cellular debris located in the periphery of vessel wall. The most common lesion detected was grade 1 (9 ilea and 4 brains). We did not find apoptotic nuclear changes in SMCs or apoptotic inclusion bodies within resident cells. Our study indicates, that (1) Stx produced during edema disease does not cause SMC apoptosis 14-15 dpi; (2) SMCs undergo an array of changes from degeneration to necrosis.

Localization of potential binding sites for the edema disease verotoxin (VT2e) in pigs.

The purpose of this study was to identify organs and cells to which the edema disease verotoxin (VT2e) could bind in pigs. Frozen 4-5 microns thick sections of organs usually affected in edema disease (colon, spinal cord, cerebellum and eyelid) and organs not usually affected (liver, ileum) from two 5- to 6-week-old weaned pigs were permeabilized with acetone, then exposed to VT2e. Unbound VT2e was removed by washing and bound VT2e was detected by immunohistochemistry. In the eyelid, double-label immunofluorescence was used to identify the cells to which VT2e bound. VT2e was shown to bind to all six organs that were examined. The toxin bound to arteries in all organs, to veins in all organs except the liver, and to enterocytes in the ileal crypts. Double labelling of eyelid with monoclonal antibodies specific for von Willebrand factor or alpha-smooth actin and VT2e showed that the toxin bound to endothelial and vascular smooth muscle cells. The binding of VT2e to endothelium is consistent with findings for other verotoxins but binding to vascular smooth muscle has not been reported for other verotoxins. It is concluded that i) factors other than the presence of receptors for VT2e influence the development of lesions in edema disease, and ii) smooth muscle necrosis, which is characteristic of the vascular lesions in edema disease, may be due to a direct action of toxin on smooth muscle cells.

Neonatal Escherichia coli infection and segmental arterial necrosis: similarity to edema disease of weanling swine.

A 32-wk gestation female patient had Escherichia coli pneumonia, hyaline membranes, and pulmonary hemorrhage and died 20 h after birth. E. coli was cultured from the placenta and from both lungs at autopsy. In the lungs and other organs, bland segmental necrosis of the wall of small arteries and arterioles was observed. It was morphologically indistinguishable from that seen in naturally occurring and experimentally induced edema disease of swine, which suggests both conditions may share a common pathogenesis. In swine, the disease is caused by Shiga-like enterotoxin-producing E. coli. To our knowledge, this is the first documentation of edema disease-like arterial lesions in humans.

An experimental model for subclinical edema disease (Escherichia coli enterotoxemia) manifest as vascular necrosis in pigs.

An experimental model for subclinical edema disease was developed in weanling pigs. In multiple experiments, 3-week-old pigs were weaned, then inoculated intragastrically with 10(10) colony-forming units of an SLT-IIv-positive strain of Escherichia coli originally isolated from a pig with edema disease (principals). Control pigs were inoculated with a nonpathogenic E coli strain. Of 39 principals, 8 developed clinical edema disease within 14 days after inoculation. However, 20 of 21 principals that did not develop clinical signs of edema disease, but were submitted for necropsy examination at 14 days after inoculation, had characteristic vascular lesions of edema disease. Vascular lesions, found principally in ileum and brain, consisted of segmental necrosis of myocytes in the tunica media of small arteries and arterioles. None of the pigs inoculated with a nonpathogenic strain of E coli developed edema disease or vascular lesions. None of the principals necropsied at 2 days after inoculation had vascular lesions. Development of vascular lesions by 14 days after inoculation was used as the end point for detecting subclinical edema disease in the model.

Reproduction of edema disease of swine with purified Shiga-like toxin-II variant.

Twenty-one weaned Yorkshire-Landrace pigs were injected intravenously with graded doses of purified Shiga-like toxin-II variant (edema disease toxin). In a preliminary study, three pigs (Nos. 1, 2, 3) were injected with 48, 24, and 12 ng, respectively, of SLT-IIv/kg of body weight. Subsequently, three groups (Nos. 4, 5, 6) of six pigs each were injected with 6, 3, and 1.5 ng, respectively, of SLT-IIv/kg of body weight. Severe clinical signs and histologic lesions characteristic of edema disease developed in pigs Nos. 1, 2, and 3, and all six pigs in group No. 4. Eight of these pigs were euthanatized in extremis (mean time to death was 34 hours) and one died of the disease (52 hours). Moderate signs and lesions of edema disease were observed in all pigs in group No. 5, and three pigs were euthanatized (mean time to euthanasia was 42 hours). Mild signs and lesions were observed in three pigs in group No. 6. The most common gross pathologic changes were edema of the eyelids, submucosa of the stomach, and mesentery of the spiral colon and hemorrhage of the colon and cerebellum. Microscopic lesions were associated with vascular injury and included vessel necrosis, perivascular edema and hemorrhage, and superficial colonic and cecal erosions. The vascular lesions were observed in the cerebellar folia, submucosa and mucosa of the stomach, cecum, colon, and sporadically in the retina. None of the clinical signs associated with endotoxin were observed.

Effects of Escherichia coli Shiga-like toxins (verotoxins) in pigs.

Escherichia coli K12 strains TB1(pCG5), with the genes for Shiga-like toxin IIv from an edema disease isolate of E. coli and TB1(pCG5-1), with the toxin genes inactivated by transposon mutagenesis, were used to test the hypothesis that Shiga-like toxin IIv was the same as edema disease principle. Ammonium sulfate precipitated culture supernatants from the pair of E. coli K12 strains and from a wild edema disease isolate of E. coli (E145) were tested for their ability to induce signs and lesions of edema disease in intravenously inoculated weaned pigs. Similar preparations from E. coli which produce Shiga-like toxins I and II were also tested. Preparations from E. coli TB1 (pCG5) and E145 contained high levels of Shiga-like toxin IIv and induced signs and lesions similar to those seen in edema disease, whereas preparations from E. coli TB1 (pCG5-1) failed to induce signs or lesions of edema disease. All Shiga-like toxin preparations produced delayed neurological signs, fibrinoid necrosis of arterioles and hemorrhages in the cerebellum of pigs. High doses of Shiga-like toxin IIv were associated with superficial necrosis of the colonic epithelium and vasculitis. Shiga-like toxins I and II resulted in kidney lesions but no enteric pathology. Shiga-like toxin II preparations had the lowest median lethal dose for pigs, Shiga-like toxin IIv was intermediate and Shiga-like toxin I was the least toxic.

Edema disease-like brain lesions in gnotobiotic piglets infected with Escherichia coli serotype O157:H7.

Gnotobiotic piglets inoculated with Escherichia coli serotype O157:H7 strains that produced Shiga-like toxin II developed brain lesions similar to those observed in edema disease of swine, including arteriolar necrosis and malacia. Loss of ability to produce Shiga-like toxin II resulted in loss of ability to cause brain lesions.

Ultrastructure of the intestinal mucosa in pigs experimentally inoculated with an edema disease-producing strain of Escherichia coli (0139:K12:H1).

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.

Bacterial colonization and morphology of the intestine in porcine Escherichia coli enterotoxemia (edema disease).

Twenty-one pigs were divided into three groups. Pigs in one group were inoculated with the intestinal contents which included bacteria from a pig with edema disease. Pigs in another group were inoculated with a culture of Escherichia coli serogroup O 139:K12(B):H1 isolated from the aforementioned contents, and pigs in a third group served as uninoculated controls. The infection was similar following both inocula. Enterotoxemia developed in 11 of the 14 pigs allowed to survive for more than two days. The onset varied from two to seven days after inoculation. There were maximal viable counts of E. coli in the intestine from the second day post-inoculation and thereafter. In frozen and paraffin sections, as well as by scanning electron microscopy, the organisms were seen on the surface of the small intestinal epithelium where they formed either isolated colonies or continuous layers. They colonized the lower small intestine more intensely than the upper section. The intestinal epithelium and the villi of infected pigs were indistinguishable morphologically from the tissues of three uninoculated control pigs. The diarrhea which was observed in controls and inoculated pigs before inoculation and the villus atrophy in controls and inoculated pigs indicated a preexisting infection with at least one other agent.

Effects of continuous intravenous infusion of Escherichia coli endotoxin into swine.

Escherichia coli endotoxin was continuously infused IV into 31 pigs. The response was related to the infusion rate. Of 13 pigs given endotoxin at the rate of 15.4 and 23.1 micrograms/kg of body weight/hour, 10 had bilateral renal cortical necrosis similar to a generalized Shwartzman lesion. All 13 pigs given endotoxin at the rate of 6.6 to 12.1 micrograms/kg/hour survived the infusion and were euthanatized 7 to 12 days later. Most of the pigs that developed chronic neurologic disturbances were given the smaller dose of endotoxin. Of 5 pigs given endotoxin at the rate of 22.0 to 23.1 micrograms/kg/hour after receiving 200 U of sodium heparin/kg of body weight, 1 died during the 48-hour period of infusion and the 4 pigs euthanatized 8 to 12 days later had lesions that were similar to those produced by endotoxin without heparin. The 4 control pigs given continuous IV infusion of isotonic saline solution at a rate of 30 dl/hour had no clinical signs or pathologic lesions. The continuous IV infusion of endotoxin into pigs at various doses produced similar clinical signs of endotoxic shock. Pigs that died acutely had widespread thrombosis. The hematologic findings indicated an intravascular coagulopathy and some of the clinical signs and lesions in the pigs resembled changes in the CNS in pigs with spontaneous edema disease (ED); however, distinct differences were also observed. Fibrinoid vascular necrosis did not occur in the CNS, and this lesion is consistently present in naturally occurring ED. The exact nature of the causal substance or substances causing ED is still unknown, but is considered an angiotoxin associated with hemolytic E coli.

Pathogenesis of edema disease in swine: pathologic effects of hemolysin, autolysate, and endotoxin of Escherichia coli (O141).

Hemolysin, cell-free autolysate, and lipopolysaccharide (LPS) prepared from Escherichia coli (O141) were parenterally administered to 113 weaned pigs. Both the hemolysin and the cell-free autolysate were crude preparations which probably contained several biologically active substances. Pigs in all groups which die less than 72 hours after injection had similar gross and microscopic lesions. The pigs which survived (chronically affected pigs) were killed 3 to 12 days after injection. Of the pigs that lived more than 72 hours after injection, those given hemolysin and autolysate had generalized vascular myolysis and fibrinoid necrosis, whereas those given LPS had morphologically normal blood vessels. The vascular changes produced by hemolysin and autolysates of E coli (O141) were the same as the histologic angiopathy of naturally occurring edema disease of pigs. The LPS produced acute lesions of endotoxin shock in the pigs, but did not produce the angiopathy characteristic of edema disease. Typical clinical signs of naturally occurring edema disease were not a consistent observation in any of the treatment groups.

[Estimation of pathomorphological and histochemical changes in the liver of pigs in the course of edema disease of swine]. / Ocena zmian patomorfologicznych i histochemicznych watroby w przebeigu choroby obrzekowej swin

Post-mortem examinations concerned 16 pigs which had died of the oedema disease. A group of 8 healthy pigs were slaughtered at the Lublin abattoir and used as controls. All animals were subjected to detailed autopsy, and sections of the liver were taken for histological examination. The fixed material was cut into sections by the paraffin and refrigeration methods. To the routine staining with haematoxylin and eosin, colour reactions were added to reveal glycogen, simple fats and acid and alkaline phosphatase. The macro- and microscopic examinations demonstrated chronic, focal inflammation of the liver capsule connected with liver cirrhosis. Hyperaemia of the liver was noticed in all cases. A decrease of glycogen, associated with progressive fatty infiltration of the liver cells, was observed in the diseased pigs in contrast to the control group. An increase of the activity of hydrolytic enzymes and a positive reaction to cholesterol compounds are symptoms of progressive catabolic changes in the liver cells.