To reduce the need for antibiotics in animal production, alternative approaches are needed to control infection. We hypothesized that overexpression of native defensin genes will provide food animals with enhanced resistance to bacterial infections. In this study, recombinant porcine beta-defensin 2 (PBD-2) was overexpressed in stably transfected PK-15 porcine kidney cells. PBD-2 antibacterial activities against Actinobacillus pleuropneumoniae, an important respiratory pathogen causing porcine contagious pleuropneumonia, were evaluated on agar plates. Transgenic pigs constitutively overexpressing PBD-2 were produced by a somatic cell cloning method, and their resistance to bacterial infection was evaluated by direct or cohabitation infection with A. pleuropneumoniae. Recombinant PBD-2 peptide that was overexpressed in the PK-15 cells showed antibacterial activity against A. pleuropneumoniae. PBD-2 was overexpressed in the heart, liver, spleen, lungs, kidneys, and jejunum of the transgenic pigs, which showed significantly lower bacterial loads in the lungs and reduced lung lesions after direct or cohabitation infection with A. pleuropneumoniae. The results demonstrate that transgenic overexpression of PBD-2 in pigs confers enhanced resistance against A. pleuropneumoniae infection.T he availability of antibiotics for treating bacterial infection has significantly improved the health of animals and humans. Administration of antibiotics at low doses to food animals has been practiced to promote better health and animal performance in many regions (1). To reduce the development of antimicrobial resistance, approaches to decrease antibiotic use in animal production are needed.Antimicrobial peptides (AMPs), a family of short cationic amphiphilic peptides with antimicrobial and immune modulation activities, exist in nearly every life form as natural anti-infective therapeutic agents (2). Some AMP genes have been used to generate transgenic (TG) mice to enhance resistance to bacterial infection. For example, expression of a synthetic cecropin-class lytic peptide in TG mice displayed enhanced resistance to Brucella abortus (3). Expression of an additional porcine cathelicidin peptide, PR-39, in mice showed increased resistance to group A Streptococcus infection (4). Furthermore, enhanced resistance to Actinobacillus suis infection was observed in proptegrin-1 TG mice (5). These studies demonstrate that AMPs potentially can lead to the development of infection-resistant animals, at least in TG mouse models. However, few reports are available on large food animals such as pigs.Defensins comprise a major family of AMPs, playing an important role in innate immunity. Porcine beta-defensin 2 (PBD-2) is thought to be an important AMP. PBD-2 provides a first line of defense against bacterial infection in pigs, because this AMP is highly expressed in epithelial cells (6, 7); moreover, PBD-2 demonstrates excellent antimicrobial activity with a broad spectrum but without hemolytic activity under physiological conditions (8).In the c...