A technique for inducing B-cell ablation in chickens by in ovo injection of cyclophosphamide

The effect of cyclophosphamide (CY) treatment in ovo on avian B and T cells was studied. CY was injected in ovo on the 16th, 17th, and 18th days of in cubation. Blood samples were collected periodically from CY-treated and non treated birds after hatch and were used to measure blood lymphocyte respons es to the T-cell and B-cell mitogens, concanavalin A and lipopolysaccharide (LPS), respectively. Additionally, flow cytometric analysis was used to de termine the presence of B and T cells in peripheral blood, and birds were v accinated with Newcastle disease virus (NDV) antigen at 3 wk of age and boo ster vaccinated at 5 wk of age. CY treatment reduced hatchability by 35%-40 %, increased mortality by 3%-5% within the first 2 wk of life, and induced a significant retardation in body weight gains. At 2 wk of age, approximate ly 50% of CY-treated birds were devoid of B-cell mitogenic responsiveness w hile demonstrating significant T-cell mitogenic responsiveness. However, B- cel; responses were observed at 4 and 6 wk from a small percentage of birds that were originally T-cell responsive and B-cell nonresponsive at 2 wk of age. Flow cytometric analysis of peripheral blood lymphocytes revealed tha t CY-treated birds had significantly less B-cells (or were devoid of B cell s) than the corresponding nontreated control birds. However, no significant difference in the T-cell percentage was observed between CY-treated and no ntreated birds. CY-treated birds did not produce detectable antibodies specific for NDV dur ing the first and second weeks postvaccination, as demonstrated by hemagglu tination inhibition assay. However, antibodies were detected in some CY-tre ated birds 10 days postbooster. Those antibody-positive birds were found to be the same birds that had subsequently responded to the LPS mitogen on th e blastogenesis microassay. This study indicates the importance of monitori ng the B- and T-cell responses in CY-treated birds to identify those birds in which B-cell regeneration may have occurred.