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Hemolytic-uremic syndrome (HUS) is the leading cause of acute renal failure in young children (13-16). Strong epidemiological evidence links the majority of HUS cases in children to infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) (1, 7, 12, 21). STEC strains liberate one or both of two toxins, known as Stx1 and Stx2 (16, 17), of which Stx2 appears to be the one most frequently linked with HUS (15). Stx-related HUS tends to occur either sporadically (14) or in outbreaks that can be traced to a common source of bacterial contamination (10). Incidents are more frequent in certain geographic locations and at certain times of the year. Although there are many STEC serotypes (14, 15, 21), O157:H7 is the one most frequently linked to HUS in children and the very elderly in the United States. Watery, mostly bloody diarrhea is the predominant symptom. Following a prodromal period of several days, HUS and other systemic complications may develop in certain individuals. HUS is marked by microangiopathic hemolytic anemia, thrombocytopenia, renal dysfunction, and on rare occasions neurological complications (9, 25).

Currently there is no effective treatment or prophylaxis for HUS. Stx appears to induce little serum antibody even in recently confirmed cases of HUS (3, 15), and the use of human immunoglobulins in children at risk has had little impact on the clinical outcome (3). In general, passively administered specific antibodies have been much more effective in preventing toxin-mediated diseases than in protecting against microbial agents. Antibodies against tetanus represent a good example. Therefore, we believe that exogenously produced and administered neutralizing antibodies will have a greater impact on the outcome of HUS if administered early in the course of the infection. The prodromal period between onset of diarrhea and development of HUS provides a window for early intervention which may improve the clinical outcome. Administration of antitoxin antibody will likely prevent HUS in contact cases. The purpose of this study was to determine whether Stx2 antibody administration could prevent systemic complications associated with Stx2 absorption from the gut as in children with HUS. Gnotobiotic (GB) piglets have been shown to be highly susceptible to infections with enterohemorrhagic E. coli. When challenged orally, GB piglets exhibit profound diarrhea due to severe mucosal damage associated with bacterial attachment and effacement of colonocytes (22, 23). More than 85% of GB piglets infected with STEC strains develop toxin-mediated neurological lesions manifested clinically by ataxia, head-pressing, recumbency, and death (24). Like humans, GB piglets develop complications when the infecting STEC strain produces Stx2; the only difference is that humans develop HUS and piglets develop neurological complications. In this study we used the GB piglet model to determine whether exogenous, Stx2-specific neutralizing antibody, administered at intervals following oral challenge with a Stx2-producing strain, could protect piglets against neurological complications.

Bacterial strains. E. coli O157:H7 strain 86-24, which produces Stx2 only, was used in these experiments (9). Strain TUV86-2, a Stx2 deletion mutant (11), was included to illustrate the direct link between Stx2 and central nervous system (CNS) involvement in piglets. Bacteria were grown in LB broth MacConkey (Difco Laboratories, Detroit, Mich.). Bacteria from infected pig tissue were cultured at 37°C on MacConkey (Difco) and blood agar plates (Becton Dickinson Microbiology Systems, Cockeysville, Md.). Antiserum was produced by immunizing two piglets six times over 6 weeks with intramuscular injections of 200 µg of affinity-purified Stx2 (5), suspended in 1 ml of phosphate-buffered saline (PBS), and emulsified with an equal volume of Freund's incomplete adjuvant. Stx2 toxoid (formalin inactivated) was used for the first two injections; these were followed by four injections of active Stx2 toxin.

Animals and experimental procedure. Fifty-nine GB piglets, derived by cesarean section from five litters, were randomized into five groups (Table 1) and maintained within sterile isolators for the duration of the experiment. Within 24 h of birth, piglets were challenged orally with 10¹⁰ E. coli O157:H7 strain 86-24. This high inoculum usually induces, within 48 to 96 h of challenge, neurological signs and brain lesions associated with Stx2 in >85% of piglets. Thirty piglets were treated with a single intraperitoneal (i.p.) injection of 5 ml (~4 ml/kg of body weight) of swine Stx2 antiserum, given at 6 (8 animals), 12 (11 animals), or 24 (11 animals) h after bacterial challenge. Sixteen control piglets were given a single i.p. injection of PBS (6 animals) or 5 ml (~4 ml/kg) of control serum (10 animals) 6 and 12 h after bacterial challenge, respectively. Two additional control groups were also included; a group of five untreated animals was challenged with the Stx2 deletion mutant strain TUV86-2, and a second group of four was neither challenged nor treated (Table 1). After euthanasia, piglets were examined for gross abnormalities. Liver, kidney, small and large intestine, and brain tissue were formalin fixed for histopathology. Blood and small and large intestinal mucosal scrapings were streaked on blood and MacConkey agar plates for bacterial identification and quantitation.

Clinical observations. Two piglets that succumbed within 48 h after bacterial challenge from severe diarrhea, before neurological symptoms were apparent, and two that died overnight and therefore could not be assessed clinically or histologically were excluded from the study. The remaining 51 challenged piglets developed various degrees of diarrhea and wasting which did not appear to have been influenced by the treatment. Of the 30 animals that received Stx2 antiserum, 4 developed neurological abnormalities (Table 1), including ataxia, incoordination, head pressing, and finally recumbency and leg paddling. Progression of symptoms was rapid and occurred within 4 to 8 h of onset. No symptoms or lesions were observed in eight animals treated 6 h after bacterial challenge. Of the 11 animals treated 12 h after bacterial challenge, one developed mild nonfatal neurological symptoms. Of the 11 animals treated 24 h after challenge, 3 developed symptoms (Table 1). Five of the six placebo-treated piglets developed neurological symptoms, while 8 of the 10 treated with swine control serum developed symptoms. The results show a clear time-related response. Four age-matched animals, which were neither treated nor challenged with bacteria, remained healthy throughout the experiment. None of the five piglets challenged with Stx2 deletion mutant strain TUV86-2 showed neurologic symptoms or lesions, but all had diarrhea.

View larger version (157K): [in this window] [in a new window]   FIG. 1.   Section through the large intestine of a piglet challenged orally with E. coli O157:H7 strain 86-24 4 days earlier. A disrupted colonic surface damaged by bacterial attachment and effacement seen in the middle, with no evidence of inflammation. The mucosa on either side of the lesion looks still intact (hematoxylin and eosin; original magnification, ×400).

View larger version (150K): [in this window] [in a new window]   FIG. 2.   Section through the cerebellum of a piglet challenged orally with E. coli O157:H7 strain 86-24, which produces Stx2, showing focal hemorrhages (arrows) in the molecular and granular layers (hematoxylin and eosin; original magnification, ×400).