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

Abstract

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.

FIG. 1.

Box plot depiction of daily fecal and blood verocytotoxin titers in pigs inoculated with Stx2e-producing E. coli. Fifty pigs were assayed for verocytotoxin in the RBC fraction of their blood (35 subclinical pigs; 15 clinical pigs on days 2 to 3 p.i., 15 on days 4 to 5 p.i., 8 on days 6 to 7 p.i., and 3 on days 8 to 9 p.i.). Blood samples were collected from each pig every other day from day 2 to 10 or day 3 to 11 p.i. Thirty pigs were assayed for fecal Stx2e on the same days (21 subclinical pigs; 9 clinical pigs on days 2 to 3 p.i., 8 on days 4 to 5 p.i., and 4 on days 6 to 7 p.i.). Horizontal lines inside boxes represent medians. The lower and upper borders of the boxes represent the 25th and 75th percentiles, respectively. Error bars indicate lowest and highest values (range). Some lines overlap.

FIG. 2.

Log₂ verocytotoxin titers in the RBC fractions of clinical (n = 15) and selected subclinical (n = 5) pigs inoculated with Stx2e-producing E. coli. All five subclinical pigs had at least one blood sample positive for verocytotoxin. A sample was considered to be positive for Stx2e if the Vero cell titer was ≥6 log₂ units and the sample (or another sample of the same type from the same pig) was neutralized by a bovine polyclonal antibody against Stx2e but was not neutralized by fetal calf serum. The line indicates the maximum nonspecific verocytotoxicity of blood.

FIG. 3.

Extent of vascular necrosis (percent necrotic arteriolar profiles per tissue section) in relation to peak verocytotoxin titer in subclinical pigs inoculated with STEC (n = 25). Samples were considered to be positive for Stx2e if the Vero cell titer was ≥6 log₂ units and the sample (or another sample of the same type from the same pig) was neutralized by a bovine polyclonal antibody against Stx2e but was not neutralized by fetal calf serum. The line indicates the maximum nonspecific verocytotoxicity of blood.

FIG. 4.

FACS analysis of Stx2e binding to porcine RBCs in vitro. Three to four samples per pig (collected over a period of 2 months at 1- to 3-week intervals) were analyzed and results were averaged. This analysis was performed for 5 pigs with the LB phenotype (MFI range, 5 to 12) and 29 pigs with the HB phenotype (MFI range, 18 to 37). Bars, standard errors of the means.