Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347 245 to be used successfully in the immuno-diagnosis of IBV and in immunization against this virus. Projeto Financiado pela Fundac ¸ ão de Auxílio à Pesquisa do Estado de São Paulo (FAPESP), processos no 02/08649-1 e no 01/14950-3 e bolsa de estudos concedida pela CAPES. doi:10.1016/j.vetimm.2008.10.074 Construction and expression of scFv antibody fragment specific for infectious bronchitis virus in Escherichia coli Aline G. Caetano * , Aliandra M. Gibertoni, Mariana C.M. Gonc ¸ alves, Hélio J. Montassier * Laboratório de Virologia e Imunologia, Departamento de Microbiologia – Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Rod. Prof. Paulo D. Castellane, s/n. Jaboticabal, SP, Brazil Keywords: Infectious bronchitis virus; Monoclonal scFv antibody; Recombinant antibody; Escherichia coli E-mail addresses: alinegcaetano@yahoo.com.br (A.G. Cae- tano), heliojm@fcav.unesp.br (H.J. Montassier). Species: Avian A Bronchitis Virus (IBV) was produced at high level in Escherichia coli. The codifying genes functional single-chain Fv antibody fragment (scFv) specific for the H120 vaccine strain of Infectious for variable regions of immunoglobulin (Ig) heavy chain (VH) and light chain (VL) were ampli- fied from spleen RNA extracts collected from SPF chickens immunized against IBV, using two specific pair of primers by RT-PCR and these amplicons were connected to a flexible oligo-peptide linker, using another specific set of primers and PCR. The final construct of this recombinant gene (VH-linker-VL), which is able to codify the ScFv antibody fragments, was inserted into a phagemid pCANTAB 5E followed by panning – selection with the Recombinant Phage Antibody System (RPAS) against purified IBV parti- cles. The scFv gene inserted into the recombinant phagemid pCANTAB 5E–H11 and harvested from a positive clone car- rying anti-IBV specificity was subcloned into pET28a fused to N-terminal His-tag sequence in frame and, following IPTG induction, it was overexpressed in E. coli BL21. The presence of scFv anti-IBV antibodies was determined by sodium dodecyl sulfate-polyacrylamide gel electrophore- sis (SDS-PAGE) followed by Western blotting analyses. A protein fraction with the expected molecular weight of approximately 32 KDa and carrying the poly-histidine tag was recognized by anti-His 6 MAb. This monoclonal scFv antibody produced here combined specifically with the subunit 1 of spike glycoprotein (S1) of IBV and did not react with other virus structural proteins. Thus, such recombi- nant antibody fragment could be useful to characterize more precisely the conformational structure of S1 glycopro- tein and its immunogenicity, as well as its variability, since this protein carries the major virus-neutralizing epitopes and suffers preferentially more variability during virus evo- lution. Projeto Financiado pela Fundac ¸ ão de Auxílio à Pesquisa do Estado de São Paulo (FAPESP), processos no 02/08649- 1 e no 01/14950-3 e bolsa de estudos concedida pela CAPES. doi:10.1016/j.vetimm.2008.10.075 TLR7 and iNOS RNA expression in spleen cells of coconut meal-fed broilers after a viral challenge with IBVD Susan D. Eicher 1,* , Tsang L. Lin 2 , Ching C. Wu 2 , Todd J. Applegate 3 , John A. Patterson 3 1 USDA-ARS, Purdue University, West Lafayette, IN, USA 2 Department of Comparative Pathobiology, Purdue Univer- sity, West Lafayette, IN, USA 3 School of Veterinary Medicine and Department of Animal Science, Purdue University, West Lafayette, IN, USA Keywords: TLR7; iNOS; Coconut meal; IBVD E-mail address: spruiett@purdue.edu (S.D. Eicher). Species: Avian Coconut meal is immunomodulating in part because of its lauric acid content. Whether this dietary immunomoulator can enhance innate immunity of chick- ens to circumvent or combat a viral challenge has not been studied. Chickens are frequently raised in confinement buildings, but a growing trend for pasture raised birds is evident. The objective of this work was to determine the effect of coconut meal on innate immune function of pasture and confinement raised chickens without and with Infectious Bursal Disease (IBDV). Sixty day-old broiler chickens were assigned to either a coconut meal diet or a standard corn/soy diet containing antibiotics in each of two studies. The first study was conducted with pasture raised broilers and the second with broilers in typical confinement housing. After receiving the diets for 4 weeks, birds were challenged orally with 4.1 × 10 3 EID50 IBDV virus. Six chickens from each dietary group were euthanized at 0, 24 and 96 h after challenge and spleens harvested for determination of toll-like receptor (TLR) 7 and inducible nitric oxide synthase (iNOS) RNA expression. Toll-like receptor 7 increased (P < 0.05) following the viral challenge in both pasture raised and confinement raised chickens and was greater (P < 0.05) for the coconut diet at 96 h. The expression of iNOS in the spleen cells did not change in the confinement raised birds (P >0.10), but was initially greater in the pasture raised birds, suggesting exposure to bacterial pathogens in the environment. iNOS decreased by 1.67-fold by 96 h compared to 0 h for birds fed the corn/soy diet and decreased by more than 10,000-fold for coconut meal fed birds. iNOS was less (P < 0.05) in the birds fed the coconut diet than for the corn/soy fed birds at 96 h post challenge. These data show the viral challenge enhanced TLR7 expression by 96 h after challenge and was affected by dietary treatment by that time. Expression of iNOS was variably affected by the viral challenge in birds raised under different conditions and fed different diets. These results will be useful for producers in determining the risks and benefits