Evaluation of inhibition of Eimeria tenella sporozoites by antibody fragments expressed in pea

Coccidiosis in chicken causes great economic losses. Increasing resistance of Eimeria species to anticoccidials has forced the search for alternative methods of control. The present study evaluates the anticoccidial activity of some anti-Eimeria tenella antibody fragments expressed in pea plants. Both in vitro and in vivo infection assays including indirect immunofluorescence, in vivo evaluation of antibody neutralization and cell culture invasion-inhibition assays were used to study the inhibitory effect of these antibody fragments on E. tenella sporozoites. Seven of nine antibody fragments (Ab1, Ab4, Ab5, Ab6, Ab7, Ab8 and Ab9) showed binding to sporozoites of E. tenella in an indirect immunofluorescence test. Only two antibodies (Ab4 and Ab5) cross reacted with sporozoites of E. maxima, E. acervulina and E. brunetti. The localization of specific fluorescence differed between species. Ab binding with sporozoites was seen in the area of both anterior and posterior refractile bodies in case of E. tenella, E. brunetti, and E. maxima but was only observed in the posterior refractile body in case of E. acervulina. No antibody binding was observed on merozoites. The suitability of antibody fragments to alter the infectivity of E. tenella sporozoites to Madin Darby Bovine Kidney cells (MDBK) was examined in vitro and the invasion-inhibition rates were quantified by flow cytometry. To assess the inhibitory effect on parasite reproduction, the in vivo antibody neutralization assay was done by retrograde infection of chicken with sporozoites previously incubated with antibody fragments. In vitro invasion rates were reduced by incubation with antibody fragments by approximately 24 to 45 %, with Ab6 and Ab7 showing the most distinct effect. However, proliferation rates (PR) of the respective MDBK cultures were also clearly reduced by 15 to 26 %. PR of MDBK cells treated with 1:1000, 1:100, 1:10 and undiluted mixed antibody fragments were reduced by 1%, 10%, 16%, and 26% with a reduction of invasion rates by 0%, 9%, 15% and 18%, respectively. Immune sera reduced the invasion rates by 16% to 70% and increased PR of the host cells. It appeared that the preparations of the antibody fragments contained compounds cytotoxic to MDBK cells and thus invasion inhibition could not be unequivocally evaluated in vitro. However, after incubation with antibody fragments sporozoites displayed a reduced ability to reproduce after intracloacal application to chicken (especially Ab1, Ab3, Ab5 and Ab9). Other antibody fragments (Ab2, Ab4, Ab6, Ab7 and Ab8) were less capable to reduce sporozoite infectivity and reproduction. More investigations are still required to study the possible use of antibody fragments and their application to infected chicken exposed to coccidiosis