Recombinant Iss as a potential vaccine for avian colibacillosis.

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 microg of GST-Iss, and 1/12 chickens immunized with 50 microg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.

Early attachment sites for Shiga-toxigenic Escherichia coli O157:H7 in experimentally inoculated weaned calves.

Weaned 3- to 4-month-old calves were fasted for 48 h, inoculated with 10(10) CFU of Shiga toxin-positive Escherichia coli (STEC) O157:H7 strain 86-24 (STEC O157) or STEC O91:H21 strain B2F1 (STEC O91), Shiga toxin-negative E. coli O157:H7 strain 87-23 (Stx(-) O157), or a nonpathogenic control E. coli strain, necropsied 4 days postinoculation, and examined bacteriologically and histologically. Some calves were treated with dexamethasone (DEX) for 5 days (3 days before, on the day of, and 1 day after inoculation). STEC O157 bacteria were recovered from feces, intestines, or gall bladders of 74% (40/55) of calves 4 days after they were inoculated with STEC O157. Colon and cecum were sites from which inoculum-type bacteria were most often recovered. Histologic lesions of attaching-and-effacing (A/E) O157(+) bacteria were observed in 69% (38/55) of the STEC O157-inoculated calves. Rectum, ileocecal valve, and distal colon were sites most likely to contain A/E O157(+) bacteria. Fecal and intestinal levels of STEC O157 bacteria were significantly higher and A/E O157(+) bacteria were more common in DEX-treated calves than in nontreated calves inoculated with STEC O157. Fecal STEC O157 levels were significantly higher than Stx(-) O157, STEC O91, or control E. coli; only STEC O157 cells were recovered from tissues. Identifying the rectum, ileocecal valve, and distal colon as early STEC O157 colonization sites and finding that DEX treatment enhances the susceptibility of weaned calves to STEC O157 colonization will facilitate the identification and evaluation of interventions aimed at reducing STEC O157 infection in cattle.

Colonization of gnotobiotic piglets by a luxS mutant strain of Escherichia coli O157:H7.

Gnotobiotic piglets inoculated with Escherichia coli O157:H7, its luxS mutant derivative, or nonpathogenic E. coli were evaluated for attaching and effacing lesions. Although no differences in clinical symptoms were seen between pigs inoculated with the parent and those inoculated with the luxS mutant, the luxS mutant-inoculated pigs had a lower frequency of attaching and effacing lesions in the spiral colon than parent strain-inoculated pigs.

Long polar fimbriae contribute to colonization by Escherichia coli O157:H7 in vivo.

The contribution of long polar fimbriae to intestinal colonization by Escherichia coli O157:H7 was evaluated in sheep, conventional pigs, and gnotobiotic piglets. E. coli O157:H7 strains with lpfA1 and lpfA2 mutated were recovered in significantly lower numbers and caused fewer attachment and effacement lesions than the parent strain.

Comparative pathogenicity of Escherichia coli O157 and intimin-negative non-O157 Shiga toxin-producing E coli strains in neonatal pigs.

We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H(-) strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysin-encoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions.

Binding of shiga toxin 2e to porcine erythrocytes in vivo and in vitro.

Shiga toxin 2e (Stx2e), produced by host-adapted Shiga toxin-producing Escherichia coli (STEC) strains, causes edema disease in weaned pigs. Edema disease is manifested as vascular necrosis, edema, neurologic signs, and death. In this study we sought to determine the correlation between the presence of Stx2e in the blood of STEC-inoculated pigs and the disease outcome. Eleven of 15 (73%) pigs with clinical and 5 of 35 (14%) pigs with subclinical edema disease had detectable levels of Stx2e in the red-blood-cell (RBC) fraction of their blood but not in serum or plasma. The presence of Stx2e in the RBC fraction was strongly associated with the development of clinical disease (relative risk, 5.8; P < 0.0001). Subclinical pigs with Stx2e in their blood developed more-extensive vascular lesions than pigs without detectable Stx2e in their blood (average proportions of necrotic arterioles, 63 and 27.5%, respectively; P = 0.001). Variations in RBC-bound Stx2e levels could in part reflect variations in the binding capacity of RBCs. As an initial step toward addressing this possibility, assays were conducted to determine if pigs vary in the Stx2e binding capacity of their RBCs. An in vitro study of noninoculated pigs demonstrated two phenotypes based on the capacity of the RBCs to bind Stx2e. While RBCs from most of the pigs consistently bound high levels of Stx2e (high-binding phenotype), consistently low Stx2e binding was detected in RBCs from a few pigs (low-binding phenotype). The low- and high-binding phenotypes of individual pigs remained consistent throughout repeated samplings over 2 months.

Vaccination of pregnant dams with intimin(O157) protects suckling piglets from Escherichia coli O157:H7 infection.

Cattle are important reservoirs of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that cause disease in humans. Both dairy and beef cattle are asymptomatically and sporadically infected with EHEC. Our long-term goal is to develop an effective vaccine to prevent cattle from becoming infected and transmitting EHEC O157:H7 to humans. We used passive immunization of neonatal piglets (as a surrogate model) to determine if antibodies against EHEC O157 adhesin (intimin(O157)) inhibit EHEC colonization. Pregnant swine (dams) with serum anti-intimin titers of < or =100 were vaccinated twice with purified intimin(O157) or sham-vaccinated with sterile buffer. Intimin(O157)-specific antibody titers in colostrum and serum of dams were increased after parenteral vaccination with intimin(O157). Neonatal piglets were allowed to suckle vaccinated or sham-vaccinated dams for up to 8 h before they were inoculated with 10(6) CFU of a Shiga toxin-negative (for humane reasons) strain of EHEC O157:H7. Piglets were necropsied at 2 to 10 days after inoculation, and intestinal samples were collected for determination of bacteriological counts and histopathological analysis. Piglets that ingested colostrum containing intimin(O157)-specific antibodies from vaccinated dams, but not those nursing sham-vaccinated dams, were protected from EHEC O157:H7 colonization and intestinal damage. These results establish intimin(O157) as a viable candidate for an EHEC O157:H7 antitransmission vaccine.

Intimin facilitates colonization by Escherichia coli O157:H7 in adult ruminants.

We compared the magnitude and duration of fecal shedding of wild-type Escherichia coli O157:H7 to that of an isogenic intimin mutant in young adult cattle and sheep. In both ruminant species, wild-type E. coli O157:H7 was shed in greater numbers and for a longer duration than was the intimin mutant.

Bovine Spongiform Encephalopathy: Hypothetical Risk of Emergence as a Zoonotic Foodborne Epidemic.

Bovine spongiform encephalopathy (BSE) is a fatal neurological disease of cattle, recognized in Great Britain in 1986. Cases in other countries have been attributed to imports from Great Britain. The disease has not occurred in the U.S. BSE is one of a group of diseases (other examples are scrapie of sheep and Creutzfeld-Jacob disease of humans) referred to as prion diseases or transmissible spongiform encephalopathies. Under some circumstances prion diseases can be transmitted by injection or by feeding infected (abnormal prion protein-containing) tissue to susceptible hosts. BSE was disseminated by feeding meat and bone meal containing BSE agent which was not completely inactivated by rendering. BSE is hypothesized to have emerged from scrapie via recycling of rendered by-products in cattle. There is also evidence of spontaneous feed-borne transmission of BSE to wild ruminants in zoological parks and to domestic cats. It has been hypothesized that foodborne transmission of BSE to humans has occurred or could occur. This hypothesis can neither be definitively refuted nor supported. However, it seems unlikely. In spite of hundreds of years of human exposure to scrapie, there is no evidence of transmission of scrapie to humans. Even if BSE is ultimately found to be somehow transmissible to humans, the risk of foodborne transmission appears to be low for several reasons: (i) The oral route is several orders of magnitude less sensitive than the parenteral route for transmission of prion diseases; (ii) the BSE agent is only detectable in brain, spinal cord, and intestine of infected cattle, tissues infrequently used for human food; and (iii) Great Britain (where the disease occurs) destroys and bans the use of all tissues from BSE-infected cattle as well as the brains, spinal cords, and intestinal tracts from clinically normal cattle.