Concentration of 125 Globulins in Intestinal Tissues of Immunized and Non-Immunized Chickens Infected with Eimeria acervulina

Abstract

Humoral antibody response to Eimeria infections has been well established (Rose, 1982. In The biology of the Coccidia, P. L. Long (ed.). University Park Press, Baltimore, Maryland, pp. 329-371); however, the role of these antibodies in the intestine has only been speculative. Leakage of serum components into the intestine occurs during coccidial infection, but the extent of leakage is not known because only semi-quantitative methods such as dye leakage have been used to measure it. During a primary infection, leakage occurred 3.5-7 hr after Eimeria acervulina or Eimeria praecox oocysts were given orally (Long, 1968, Parasitology 58: 691-700). Leakage was not observed at 24-48 hr post-inoculation, but does occur at 72-120 hr post-inoculation, coinciding with the eruption and multiplication of merozoites in epithelial cells. Previous results to determine permeability changes in immunized birds have been equivocal because of the lack of sensitivity of the dye leakage assay (Rose et al., 1975, Parasitology 71: 357-368). These studies did, however, reveal that the permeability change to sporozoite invasion was somewhat species-specific and that the changes were not induced by non-viable coccidial preparations. We have used a sensitive, direct method to evaluate the leakage of serum proteins into the intestine of Eimeria-immunized and non-immunized chickens, using radioiodinated (125I) chicken globulins. Serum globulins were precipitated with Na2SO4, dialyzed, and lyophilized from clarified, filtered chicken serum (Garvey et al., 1977, Methods in immunology. W. A. Benjamin, Inc., Reading, Massachusetts, pp. 218220). The lyophilized proteins were radioiodinated (125I, Amersham) (Greenwood et al., 1963, Biochemistry Journal 89: 114-123). lodinated protein was separated from free iodine by passage over a Sephadex PD 10 column. Fractions on the ascending limb of the protein peak were consolidated and diluted to 1 x 106 cpm/0.5 ml with phosphate-buffered saline (pH 7.6, 0.1 M). Three groups of 3-wk-old Leghorn chicks were used in this experiment. Group 1 (n = 9) was immunized by inoculating each bird with 5,000 sporulated oocysts of E. acervulina at 3 wk and 4 wk. Group 2 (n = 4) and group 3 (n = 4) were not immunized. Groups 1 and 2 were challenged with 1 x 107 oocysts of E. acervulina at 6 wk. Group 3 was not challenged. Three-and-one-half hr after challenge, each bird from all groups was injected intravenously with radioiodinated (1251) chicken globulins. Thirty min after injection of the 125I proteins, all birds were killed by intra-cardiac injection of sodium pentobarbital. The duodenal loop and upper jejunum were excised, cut into 6 equal sections, weighed, and placed into tubes filled with 0.85% NaCl solution. After determining the amount of radioactivity, the gut pieces were removed and radioactivity in the saline was determined. The latter measurement was taken as an indication of extravascular globulin. Four hr after challenge, there were striking differences in the amount of iodinated protein associated with the intestinal tissues (Fig. 1). Nonimmunized chickens had significantly higher