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Title:   Author  item
                                            Kehrli Jr, Marcusitem
                                            Gauger, Phillipitem
                                            Cheung, Andrewitem
                                            Baker, Amyitem
                                            Lager, Kelly
  Submitted to: International Pig Veterinary Society (IPVS)
                                    Publication Type: Proceedings
                                    Publication Acceptance Date: 3/30/2010
                                    Publication  Date: 7/18/2010
                                    Citation: Kehrli, Jr., M.E., Gauger, P.C., Cheung, A.K., Vincent, A.L., Lager, K.M. 2010. Discovery of Antinuclear Antibodies in Pigs Infected with Porcine Circovirus Type 2. In: Proceedings of the International Pig Veterinary Society Congress, July 18-21, 2010, Vancouver, Canada. p. 332.Interpretive Summary:
                                Technical Abstract: Introduction.  Porcine circovirus type 2 (PCV2) causes post-weaning-multisystemic-wasting-syndrome (PMWS), a swine disease first observed in Canada in 1991 (1).  It is characterized by general wasting, respiratory disease, jaundice and pallor in young pigs resulting in production losses and variable mortality.  Since the discovery of PCV2 in 1996, this virus has been linked to other disease syndromes (porcine dermatitis and nephropathy syndrome, reproductive failure, and enteritis) that are collectively known as porcine circovirus associated disease (PCVAD).  PCV2 is ubiquitous in swine herds around the world and endemic in most herds. The majority of pigs remain clinically unaffected whereas a small proportion display severe clinical and sometimes fatal disease. We have experimentally reproduced PMWS using germ-free piglets (2).  An unusual feature of disease pathogenesis is a delayed onset of clinical disease following virus challenge and the subsequent rapid decline in the health of diseased piglets.  Here we report evidence for development of antinuclear antibodies (ANA) in sera of germ-free pigs infected with PCV2.
    Materials and Methods.  A conserved amino acid sequence based on the published capsid protein sequence of PCV2a was used to make a synthetic peptide  and to immunize rabbits for PCV2-specific antiserum production.  
    A series of animal studies were conducted with germ-free piglets derived aseptically from crossbred sows by cesarean section (2). Piglets were housed in sterile, stainless-steel pentub isolators (4 pigs/isolator) and fed a commercially-available pasteurized milk diet. Each pig within an isolator received the same treatment, either PCV2 or a sham inoculation at about 7-10 days of age.  In comparative viral pathogenesis studies, different groups of pigs were similarly challenged with swine influenza virus.
    ANA were measured by two independent methods: 1) a fluorescent microscope slide-based indirect immunofluorescence assay (IFA) (SCIMEDX HEp2 ANA IFA, Denville, NJ) and an ANA ELISA (QUANTA Lite ANA, INOVA Diagnostics, Inc., San Diego, CA) modified by using an anti-swine IgG secondary antibody (KPL, Inc., Gaithersburg, MD).
    Results.  To investigate PCV2 replication in tissue culture, a conserved peptide of the PCV2 nucleocapsid was produced to make polyclonal antisera in a rabbit.  Initial testing with this antiserum revealed significant background staining of a porcine kidney cell line (PK-15) used to propagate PCV2 in our laboratory.  The staining was most prominent in the nuclear region of PK-15 cells and was present regardless of the PCV2 infection status of the cells, suggesting the antisera was recognizing nuclear antigens unrelated to PCV2.  This led us to investigate the presence of ANA in piglet sera from prior germ-free isolator studies with PCV2 and other viruses.
    Thirty-eight pigs used in 3 separate studies were found to be free of ANA on the day of inoculation with virus.  In these studies, 21 of 38 piglets infected with PCV2 isolates developed detectable ANA of the IgG isotype based on the Hep-2 IFA ANA test.  Most piglets that developed ANA did so by 18-25 dpi with PCV2. In contrast, 7 piglets infected in 2 separate studies with a swine influenza virus remained free of detectable ANA.  To confirm these semiquantitative findings, a subset of samples from one germ-free study were tested by an ELISA that collectively detects total ANA against chromatin (dsDNA and histones), Sm/RNP, SS-A, SS-B, Scl-70, centromere and PCNA, as well as antibodies against cytoplasmic antigens such as Jo-1, mitochondria (M-2) and ribosomal-P protein. ELISA results demonstrated development of increasing levels of ANA over the course of a study in 9 of 12 piglets infected with PCV2 but not in 4 of 4 control virus-free piglets.  
     Discussion.  Antinuclear antibodies are directed against norma


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