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Joan K Lunney

Publications and source records attributed to Joan K Lunney.

13 recordsLinked to original sources
PubMed2006

A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region.

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.

Animals↗
PubMed2006

Gene expression profiling in Salmonella Choleraesuis-infected porcine lung using a long oligonucleotide microarray.

Understanding the transcriptional response to pathogenic bacterial infection within food animals is of fundamental and applied interest. To determine the transcriptional response to Salmonella enterica serovar Choleraesuis (SC) infection, a 13,297-oligonucleotide swine array was used to analyze RNA from control, 24-h postinoculation (hpi), and 48-hpi porcine lung tissue from pigs infected with SC. In total, 57 genes showed differential expression (p < 0.001; false discovery rate = 12%). Quantitative real-time PCR (qRT-PCR) of 61 genes was used to confirm the microarray results and to identify pathways responding to infection. Of the 33 genes identified by microarray analysis as differentially expressed, 23 were confirmed by qRT-PCR results. A novel finding was that two transglutaminase family genes (TGM1 and TGM3) showed dramatic increases in expression postinoculation; combined with several other apoptotic genes, they indicated the induction of apoptotic pathways during SC infection. A predominant T helper 1-type immune response occurred during infection, with interferon gamma (IFNG) significantly increased at 48 hpi. Genes induced by IFNs (GBP1, GBP2, C1S, C1R, MHC2TA, PSMB8, TAP1, TAP2) showed increased expression during porcine lung infection. These data represent the first thorough investigation of gene regulation pathways that control an important porcine respiratory and foodborne bacterial infection.

Animals↗
PubMed2006

Comparison of detection methods for Toxoplasma gondii in naturally and experimentally infected swine.

Results from recent serological surveys and epidemiological studies show that pigs raised in a variety of management systems can be carriers of the tissue cyst stage of Toxoplasma gondi. This parasite can be transmitted to humans through the consumption of improperly prepared pork, making detection and removal of infected swine carcasses from the food chain an important food safety issue. Several methods are available for detection of T. gondii infected swine, including serological assays, polymerase chain reaction, and animal bioassays. The aim of the present study was to compare the detection sensitivities of six of these commonly used methods for detection of T. gondii infection in tissues from naturally and experimentally infected pigs. The results indicate that a serum-based ELISA is the most sensitive method, of those tested, for detection of T. gondii infected swine.

Animals↗
PubMed2005

Summary of the animal homologue section of HLDA8.

Development of reagents against leukocyte differentiation antigens in veterinary immunology is slower compared to humans and mice. Cross-reactivity studies with monoclonal antibodies (mAb) generated against human molecules represent an excellent approach for the detection of new reagents for the minor characterised species. Three hundred seventy-seven commercially available mAb from different companies were tested for their reactivity with cells from 17 species--including non-human primates, ruminants, swine, horse, carnivores, rabbit, guinea pig, chicken and fish. In a first round of testing by flow cytometry (FCM) 182 mAb showed reactivity with atleast one of the species described above. Most of the cross-reactivity was found against non-human primate leukocytes, but also species in evolutionarily more distant from humans showed in some cases a clear staining pattern in flow cytometry (FCM). In a second round these FCM-results were confirmed by molecular analyses, by immunoprecipitation studies and analyses on transfectants. Interesting was the broad species-overlapping reactivity of mAb directed against CD9 (11 out of 17 species), CD11a (11/17), CD14 (11/17), CD18 (13/17), CD21 (7/17), CD29 (10/17), CD44 (13/17), CD45 (9/17), CD47 (10/17), and CD49d (13/17), CD61 (6/17), CD86 (7/17), CD91 (5/17), and CD172a (10/17), indicating evolutionary highly conserved epitopes on these surface molecules. Our results suggest the suitability of crossreactive mAb for the animal model studies. Moreover, these findings contribute to our understanding of the evolution of the immune system.

Animals↗
PubMed2005

Perspectives for artificial insemination and genomics to improve global swine populations.

Civilizations throughout the world continue to depend on pig meat as an important food source. Approximately 40% of the red meat consumed annually worldwide (94 million metric tons) is pig meat. Pig numbers (940 million) and consumption have increased consistent with the increasing world population (FAO 2002). In the past 50 years, research guided genetic selection and nutrition programs have had a major impact on improving carcass composition and efficiency of production in swine. The use of artificial insemination (AI) in Europe has also had a major impact on pig improvement in the past 35 years and more recently in the USA. Several scientific advances in gamete physiology and/or manipulation have been successfully utilized while others are just beginning to be applied at the production level. Semen extenders that permit the use of fresh semen for more than 5 days post-collection are largely responsible for the success of AI in pigs worldwide. Transfer of the best genetics has been enabled by use of AI with fresh semen, and to some extent, by use of AI with frozen semen over the past 25 years. Sexed semen, now a reality, has the potential for increasing the rate of genetic progress in AI programs when used in conjunction with newly developed low sperm number insemination technology. Embryo cryopreservation provides opportunities for international transport of maternal germplasm worldwide; non-surgical transfer of viable embryos in practice is nearing reality. While production of transgenic animals has been successful, the low level of efficiency in producing these animals and lack of information on multigene interactions limit the use of the technology in applied production systems. Technologies based on research in functional genomics, proteomics and cloning have significant potential, but considerable research effort will be required before they can be utilized for AI in pig production. In the past 15 years, there has been a coordinated worldwide scientific effort to develop the genetic linkage map of the pig with the goal of identifying pigs with genetic alleles that result in improved growth rate, carcass quality, and reproductive performance. Molecular genetic tests have been developed to select pigs with improved traits such as removal of the porcine stress (RYR1) syndrome, and selection for specific estrogen receptor (ESR) alleles. Less progress has been made in developing routine tests related to diseases. Major research in genomics is being pursued to improve the efficiency of selection for healthier pigs with disease resistance properties. The sequencing of the genome of the pig to identify new genes and unique regulatory elements holds great promise to provide new information that can be used in pig production. AI, in vitro embryo production and embryo transfer will be the preferred means of implementing these new technologies to enhance efficiency of pig production in the future.

Animals↗
PubMed2005

Validation of a first-generation long-oligonucleotide microarray for transcriptional profiling in the pig.

A first-generation porcine oligonucleotide set, representing 13,297 cDNAs and ESTs, has been designed by Qiagen-Operon for transcriptional profiling. To validate this set, microarrays containing each 70-mer oligonucleotide, referred to as the Qiagen-NRSP8 array, were hybridized with targets from porcine adult liver, lung, muscle, or small intestine. Transcriptome analyses showed that 11,328 of the oligonucleotides demonstrated expression in at least one tissue. Statistical analyses revealed that 1810 genes showed differential expression among tissues (Bonferroni adjusted p < 0.05). Biological pathways identified by DAVID/EASE analysis using a list of 423 tissue-selective genes matched archetypal pathways in the corresponding human or mouse tissue. Real-time quantitative PCR confirmed expression patterns for 9 of 11 genes tested. Our results demonstrate that this first-generation porcine oligonucleotide array is informative and the specificity is high. This is essential validation for investigators using the Qiagen-NRSP8 array for porcine functional genomics and for using the pig in modeling important physiological problems.

Animals↗
PubMed2005

Localized multigene expression patterns support an evolving Th1/Th2-like paradigm in response to infections with Toxoplasma gondii and Ascaris suum.

Human infectious diseases have been studied in pigs because the two species have common microbial, parasitic, and zoonotic organisms, but there has been no systematic evaluation of cytokine gene expression in response to infectious agents in porcine species. In this study, pigs were inoculated with two clinically and economically important parasites, Toxoplasma gondii and Ascaris suum, and gene expression in 11 different tissues for 20 different swine Th1/Th2-related cytokines, cytokine receptors, and markers of immune activation were evaluated by real-time PCR. A generalized Th1-like pattern of gene expression was evident in pigs infected with T. gondii, along with an increased anti-inflammatory gene expression pattern during the recovery phase of the infection. In contrast, an elevated Th2-like pattern was expressed during the period of expulsion of A. suum fourth-stage larvae from the small intestine of pigs, along with low-level Th1-like and anti-inflammatory cytokine gene expression. Prototypical immune and physiological markers of infection were observed in bronchial alveolar lavage cells, small intestinal smooth muscle, and epithelial cells. This study validated the use of a robust quantitative gene expression assay to detect immune and inflammatory markers at multiple host tissue sites, enhanced the definition of two important swine diseases, and supported the use of swine as an experimental model for the study of immunity to infectious agents relevant to humans.

Animals↗
PubMed2004

Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus.

Immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine initially elicits a weak interferon (IFN)-gamma response. To improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (IL)-12 or IFN-alpha was co-administered during vaccination. In the presence of either adjuvant, at least a three-fold increase in the primary virus-specific IFN-gamma response was observed. While this enhancement was only transient (1 week) when the IL-12 expressing plasmid was used, the effect was not only still apparent at 6 weeks after vaccination in the presence of the IFN-alpha expressing plasmid but even after challenge with a virulent genetically divergent PRRSV. In contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. Despite the apparent augmentation of a T helper (Th) 1 type response by the inclusion of IFN-alpha or IL-12 during vaccination, this modulation did not necessarily correlate with a reduction in viremia. Since a similar increase in the degree of the IFN-gamma response to the PRRSV vaccine could be achieved by substituting polyinosinic-polycytidylic acid in lieu of either cytokine, exposure to PRRSV in the presence of a variety of Th 1 polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. Conceivably, such intervention could be applied to improve the formulation of anti-PRRSV vaccines.

Animals↗
PubMed2004

Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination.

The natural response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) infections and vaccinations needs to be altered so that better protection is afforded against both homologous and heterologous challenges by this pathogen. To address this problem, real-time gene expression assays were coupled with cytokine Elispot and protein analyses to assess the nature of the anti-PRRSV response of pigs immunized with modified live virus (MLV) vaccine. Although T helper 1 (Th1) immunity was elicited in all vaccinated animals, as evidenced by the genesis of PRRSV-specific interferon-gamma secreting cells (IFNG SC), the overall extent of the memory response was variable and generally weak. Peripheral blood mononuclear cells (PBMC) isolated from these pigs responded to PRRSV exposure with a limited increase in their expression of the Th1 immune markers, IFNG, tumor necrosis factor-alpha and interleukin-15 (IL15), and a reduction in the quantity of mRNAs encoding the innate and inflammatory proteins, IL1B, IL8 and IFNA. Efforts to enhance Th1 immunity, by utilizing an expression plasmid encoding porcine IFNA (pINA) as an adjuvant, resulted in a temporary increase in the frequency of PRRSV-specific IFNG SC but only minor changes overall in the expression of Th1 associated cytokine or innate immune marker mRNA by virus-stimulated PBMC. Administration of pINA, however, did correlate with decreased IL1B secretion by cultured, unstimulated PBMC but had no effect on their ability to release IFNG. Thus, while exogenous addition of IFNA during PRRSV vaccination has an impact on the development of a Th1 immune response, other alterations will be required for substantial boosting of virus-specific protection.

Adjuvants, Immunologic↗
PubMed2004

Identification of key immune mediators regulating T helper 1 responses in swine.

This publication describes the cloning of full or partial length sequences for pig TBX21 (T-bet), MYD88, ICSBP1, CD8A (CD8alpha), CD8B (CD8beta), and CD28 cDNAs. Real-time PCR assays have been developed for the relative quantitation of these products as well as previously characterized transcripts that encode exon A-containing CD45, HLX1, IRF1, STAT1 and RPL32. When used for examining temporal immune gene expression in the liver of Toxoplasma gondii infected pigs, the positive regulators of Th1 responses, IRF1, MYD88, and STAT1, were found to be expressed prior to the simultaneous upregulation of interferon gamma (IFNG), HLX1 and TBX21 gene expression. In contrast, in the mesenteric lymph node (MLN), only expression of IRF1 and IFNG was significantly upregulated. Based on their demonstrated utility in establishing an immune response pathway, these PCR assays should be valuable additions to our swine immune toolkit.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Rapid assignment of swine leukocyte antigen haplotypes in pedigreed herds using a polymerase chain reaction-based assay.

We present a simple assay to determine the swine leukocyte antigen (SLA) haplotypes of animals within two experimental populations of MHC defined miniature pigs. The Yucatan miniature pigs have four founder haplotypes ( w, x, y, z) and one recombinant haplotype ( q). The NIH miniature pigs have three founder haplotypes ( a, c, d) and two recombinant haplotypes ( f, g). Because most crossovers occur between the class I and class II regions, haplotypes can be assigned by typing one class I locus and one class II locus for practical purposes. We have previously characterized these seven founder haplotypes by sequencing the cDNA of three SLA class I loci, designated as SLA-1, SLA-3 and SLA-2 and four SLA class II loci, SLA-DQA1, SLA-DQB1, SLA-DRA1 and SLA-DRB1. These sequences were used to design allele-specific primers to amplify one MHC class I and one MHC class II gene for each haplotype. Primers were tested for specificity in homozygous and heterozygous animals. Positive control primers were also designed to amplify a portion of the E-selectin or alpha-actin gene and multiplexed with the allele-specific primers to check for false negatives. This combination of allele-specific and positive control primers produced specific and robust PCR-site-specific primer assays for assigning SLA haplotypes in the two populations.

Animals↗