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

Cyclophosphamide (CY) was injected in ovo on the 16th, 17th and 18th days of incubation. Blood samples were collected periodically from CY-treated and non-treated birds after hatch and were used to measure blood lymphocyte responses to the T-cell and B-cell mitogens, concanavalin A and lipopolysaccharide (LPS), respectively. Additionally, flow cytometric analysis was used to determine the presence of B and T cells in peripheral blood, and birds were vaccinated with Newcastle disease virus (NDV) antigen at 3 weeks of age and booster vaccinated at 5 weeks of age. CY treatment reduced hatchability by 35-40%, increased mortality by 3-5% within the first 2 weeks of life, and induced a significant retardation in body weight gains. At 2 weeks of age, approximately 50% of CY-treated birds were devoid of B-cell mitogenic responsiveness while demonstrating significant T-cell mitogenic responsiveness. However, B-cell responses were observed at 4 and 6 weeks from a small percentage of birds that were originally T-cell responsive and B-cell nonresponsive at 2 weeks of age. Flow cytometric analysis of peripheral blood lymphocytes revealed that CY-treated birds had significantly less B cells (or were devoid of B cells) than the corresponding non-treated control birds. However, no significant difference in the T-cell percentage was observed between CY-treated and nontreated birds. CY-treated birds did not produce detectable antibodies specific for NDV during the first and second weeks postvaccination, as demonstrated by haemagglutination inhibition assay. However, antibodies were detected in some CY-treated birds 10 days postbooster. Those antibody-positive birds were the same birds that had subsequently responded to the LPS mitogen on the blastogenesis microassay. It is concluded that it is important to monitor the B- and T-cell responses in CY-treated birds to identify those birds in which B-cell regeneration may have occurred.