Eight hundred Erysipelothrix strains isolated between 1992 and 2002 from swine with erysipelas in Japan were serotyped. Thirty-seven, 47, 73, and 643 strains were isolated from animals with acute septicemia, urticaria, chronic endocarditis, and chronic arthritis, respectively, of which 381, 146, 254, and 19 isolates belonged to serotypes 1a, 1b, and 2b and other serotypes, respectively. All serotype 1a isolates were further examined for acriflavine resistance and their genotypes to discriminate them from the attenuated live vaccine strain, defined as serotype 1a, which is resistant to 0.02% acriflavine and which shows low levels of pathogenicity in mice. Of the serotype 1a isolates, 64.6% were acriflavine resistant, with 98.4% of these acriflavine-resistant strains having been isolated from animals with chronic arthritis. By randomly amplified polymorphic DNA (RAPD) analysis, almost all the acriflavine-resistant serotype 1a strains showed the 253-bp band characteristic of vaccine strains and were easily discriminated from all 113 strains of acriflavine-sensitive serotype 1a strains from animals with acute and subacute swine erysipelas. The incidence of acriflavine-resistant strains of the distinctive RAPD type 1-2 was markedly higher than that of the other RAPD types and serotypes. RAPD type 1-2 strains also included a specific group identifiable by restriction fragment length polymorphism DNA analysis. Furthermore, the pathogenicities of 29 isolates of RAPD type 1-2 for mice were lower than those of the 21 isolates of other RAPD types. Our results indicate that RAPD type 1-2 strains are live vaccine strains and that 37% of the cases of chronic swine erysipelas detected in the past 11 years in Japan have occurred as a side effect of live vaccine use.

We successfully cloned and sequenced porcine toll-like receptor (TLR2) and TLR6 cDNA from porcine alveolar macrophages stimulated with 10 microg/ml lipopolysaccharide (LPS). The open reading frames (ORFs) of the porcine TLR2 and TLR6 cDNA were shown to be 2358 and 2391 bp in length and to encode 785 and 796 amino acids, respectively. The predicted amino acid sequence of porcine TLR2 was 72.3% homologous to human TLR2 and 61.0% homologous to murine TLR2. That of porcine TLR6 was 74.4% homologous to human TLR6 and 66.1% homologous to murine TLR6. Porcine TLR2 and TLR6 genes were both mapped to porcine chromosome 8 (TLR2: SSC8q21.1 --> 21.5; TLR6: SSC8p11.1 --> p21.1) by fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Western blot analysis confirmed that TLR2 and TLR6 proteins were both expressed in porcine alveolar macrophages. Further, antiporcine TLR2 and TLR6 antibodies synergistically blocked tumor necrosis factor-alpha (TNF-alpha) production by porcine alveolar macrophages stimulated with Mycoplasma hyopneumoniae. These results indicated that both TLR2 and TLR6 are important in the recognition of M. hyopneumoniae in porcine alveolar macrophages and will be useful in understanding innate immunity against M. hyopneumoniae.

ABSTRACT. A pig interleukin-21 (IL-21) cDNA was successfully cloned and sequenced from porcine peripheral blood lymphocytes (PBL) stimulated with 10 µg/ml concanavalin A (ConA), 10 µg/ml phytohemagglutinin P (PHA), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), and 0.5 µg/ml anti-porcine CD3 antibody for 48 hr. The open reading frame of the porcine IL-21 cDNA is 459 base pairs in length and encodes 152 amino acids. The predicted amino acid sequence of the porcine IL-21 shows 86.2%, 77.7%, and 58.4% identi ty to the bovine, human, and murine IL-21, respectively. The porcine IL-21 gene was mapped to porcine chromosome 8 (8q22→q23) by means of fluorescence in situ hybridization and radiation hybrid mapping, where the porcine IL-2 gene had been mapped nearby. The recombinant porcine mature IL-21 expressed by E. coli induced dose-dependent proliferation and IFN-γ production from a human NK cell line, NK0. The porcine IL-21 identified in this study will be helpful for the enhancement of innate immune responses of pigs. KEY WORDS: cloning, innate immunity, interleukin-21, NK cell, porcine.J. Vet. Med. Sci. 66(3): 269-275, 2004 Interleukin-21 (IL-21) is a novel cytokine that regulates the proliferation of T and B cells, and involves the maturation and expansion of NK cells from bone marrow progenitor cells [20]. IL-21 is produced by activated T cells [20,21], and is a four-helix-bundle type I cytokine with structural similarity to IL-2, 21]. IL-21 receptor also has significant amino acid similarities with IL-2, , and shares the common γ chain for the subunit of the IL-21 receptor complex [2]. The common γ chain-containing receptor binded by its ligand such as IL-2 and IL-21 causes the activation of the Janus tyrosine kinase family 3 (JAK3) [6,17], and transduce its signal to the downstream molecules. Recent studies have indicated that IL-21 has various biological activities that affect T and NK cell functions [20,21] . Currently, IL-21 is thought to be a mediator cytokine, which promotes the transition from innate to adaptive immunity [9,21].One of the most interesting characteristics of IL-21 is its effect on NK cell maturation and expansion [20], because pigs have high NK cell populations in the peripheral blood [31], and many piglets suffer from diarrhea and respiratory diseases in their neonatal periods during which adaptive immunity is immature. We previously reported the cloning and expression of porcine , and the IFN-γ induction by IL-18 from neonatal piglets [13]. Recently, Strengell et al. reported that IL-21 also enhances IFN-γ production from human NK and T cells in synergy with . Taken, together, we would like to isolate porcine IL-21, a recent cytokine that promotes the transition from innate to adaptive immunity [9,21], in order to utilize this cytokine for the enhancement of neonatal immunity of pigs. We also reported the cloning, and the expression of bovine IL-21 using a baculovirus expression system [15].In the present study, cDNA encoding porcine IL-21 was cloned and characterized, and the chromos...

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