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Detection of antibodies to Ovine herpesvirus-2 interleukin-10homologue in sheep-associated malignant catarrhal fever
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Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
and others identified 2 -integrins as the receptors for leukotoxin (Lkt) which is the most important virulence factor produced by Mannheimia (Pasteurella) haemolytica, the primary bacterial pathogen of bovine respiratory disease complex. By rendering an Lkt-non-susceptible murine cell-line susceptible to Lkt-induced cytolysis, we have previously demonstrated that CD18 mediates Lktinduced cytolysis. However, in that study, bovine CD18 was expressed as a heterodimer with murine CD11a which precluded the elucidation of the role of bovine CD11a. Therefore, the objective of this study was to precisely identify the role of bovine CD11a and CD18 in Lkt-binding and cytolysis of target cells. cDNA for bovine CD11a and CD18, either individually or together, was transfected into the human embryonic kidney cell-line (HEK-293) which does not express any 2 -integrins. Transfectants stably expressing monomeric CD11a, CD18, or heterodimeric LFA1 (CD11a/CD18) on their cell surface were selected by flow cytometric analysis with monoclonal antibodies specific for bovine CD11a or CD18. In Lkt-binding assays, all three transfectants, but not the parent cells, effectively bound Lkt. However, Lkt-induced cytolysis was observed only with transfectants expressing monomeric CD18 or LFA-1. Furthermore, intracellular [Ca2+ ]i elevation following exposure to Lkt, which is acknowledged as an indication of Lkt-receptor interaction, was seen only with transfectants expressing monomeric CD18 or LFA-1. Taken together, these results clearly indicate that it is the CD18 subunit that is involved in the Lkt-induced cytolysis of target cells. Although CD11a binds to Lkt, it is not involved in the events leading to intracellular [Ca2+ ]i elevation and cytolysis. The LFA-1 expression in the transfectants was stable for longer periods than either one of the subunits suggesting that association of the two subunits was necessary for their stable expression. doi:10.1016/j.vetimm.2008.10.052 Detection of antibodies to Ovine herpesvirus2 interleukin-10homologue in sheep-associated malignant catarrhal fever Robin L. Cissell 1,∗ , Shahira Abdel Wahab 2 , Robert L. Donnell 3 , Stephen A. Kania 2 1 Comparative and Experimental Medicine Program, University of Tennessee Veterinary Teaching Hospital, Knoxville, United States 2 Department of Comparative Medicine, University of Tennessee Veterinary Teaching Hospital, Knoxville, United States 3 Department of Pathobiology, University of Tennessee Veterinary Teaching Hospital, Knoxville, United States Keywords: Viral IL-10; Malignant catarrhal fever; Ovine herpesvirus-2; IL-10homologue
Species: Ruminants Interleukin-10 (IL-10) interferes with monocyte and macrophage activation of Th1 helper lymphocyte production of nitric oxide and synthesis of various inflammatory mediators. An IL-10homologue (vIL-10) is produced by several herpes viruses and is hypothesized
to help the virus down regulate, and thus evade, host immune responses. The gammaherpes rhadinovirus Ovine herpesvirus-2 (OvHV-2) encodes an IL-10 like molecule highly homologous to mammalian IL-10. This virus causes sheep-associated malignant catarrhal fever (MCF), the most common form of MCF in the United States. MCF is a lymphoproliferative and inflammatory syndrome which has delayed clinical presentation hypothesized to be influenced by the production of vIL-10. For this study, the gene encoding vIL-10, Ov2.5 ORF, was amplified by PCR, cloned, sequenced, and a predicted 30 amino acid segment from the amino terminus synthesized. This synthetic peptide was used to develop a novel direct enzyme-linked immunosorbant assay (ELISA) to detect isotype-specific antibodies to OvHV-2 vIL-10. Work to date indicates that lambs do not have detectable levels of maternally derived antibody to vIL-10 during the first few weeks of age. Ewes, which are refractory to clinical infection, generally have high levels of antibody to vIL-10. A weak correlation exists between the vIL-10 ELISA and a commercially available competitive-inhibition ELISA (CI-ELISA). We believe the vIL-10 ELISA will refine the ability to identify ruminants exposed to sheep-associated MCF, provide an important tool for determining the role of vIL-10 in disease pathogenesis, and may contribute toward the development of a new vaccine strategy for the control of malignant catarrhal fever. doi:10.1016/j.vetimm.2008.10.053 Immunoproteomic analysis of the protective outer membrane fraction of Anaplasma marginale Wendy C. Brown Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, United States Keywords: Mass spectrometry; Anaplasma marginale; Outer membranes; T lymphocytes E-mail address: [email protected]. Species: Ruminants Rickettsial pathogens in the genera Anaplasma and Ehrlichia cause acute infection in immunologically naïve hosts and are major causes of tick-borne disease in animals and humans. Immunization with Anaplasma marginale purified outer membranes induces complete protection against anaplasmosis in 75% of cattle, whereas immunization with the well-studied and immunodominant major surface proteins such as MSP2 has provided little or no protection. The completed genome sequence of A. marginale facilitated the identification of subdominant and less abundant immunogenic proteins in the outer membrane fraction, using two approaches. First, two-dimensional electrophoresis and immunoblotting of the outer membrane fraction with immune bovine sera identified numerous antigenic protein spots. Analysis of individual proteins excised from the gels by liquid chromatography and tandem mass spectrometry identified 21 novel antigens. Of particular interest is the finding that three proteins from the type IV secretion system, conju-
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
and others identified 2 -integrins as the receptors for leukotoxin (Lkt) which is the most important virulence factor produced by Mannheimia (Pasteurella) haemolytica, the primary bacterial pathogen of bovine respiratory disease complex. By rendering an Lkt-non-susceptible murine cell-line susceptible to Lkt-induced cytolysis, we have previously demonstrated that CD18 mediates Lktinduced cytolysis. However, in that study, bovine CD18 was expressed as a heterodimer with murine CD11a which precluded the elucidation of the role of bovine CD11a. Therefore, the objective of this study was to precisely identify the role of bovine CD11a and CD18 in Lkt-binding and cytolysis of target cells. cDNA for bovine CD11a and CD18, either individually or together, was transfected into the human embryonic kidney cell-line (HEK-293) which does not express any 2 -integrins. Transfectants stably expressing monomeric CD11a, CD18, or heterodimeric LFA1 (CD11a/CD18) on their cell surface were selected by flow cytometric analysis with monoclonal antibodies specific for bovine CD11a or CD18. In Lkt-binding assays, all three transfectants, but not the parent cells, effectively bound Lkt. However, Lkt-induced cytolysis was observed only with transfectants expressing monomeric CD18 or LFA-1. Furthermore, intracellular [Ca2+ ]i elevation following exposure to Lkt, which is acknowledged as an indication of Lkt-receptor interaction, was seen only with transfectants expressing monomeric CD18 or LFA-1. Taken together, these results clearly indicate that it is the CD18 subunit that is involved in the Lkt-induced cytolysis of target cells. Although CD11a binds to Lkt, it is not involved in the events leading to intracellular [Ca2+ ]i elevation and cytolysis. The LFA-1 expression in the transfectants was stable for longer periods than either one of the subunits suggesting that association of the two subunits was necessary for their stable expression. doi:10.1016/j.vetimm.2008.10.052 Detection of antibodies to Ovine herpesvirus2 interleukin-10homologue in sheep-associated malignant catarrhal fever Robin L. Cissell 1,∗ , Shahira Abdel Wahab 2 , Robert L. Donnell 3 , Stephen A. Kania 2 1 Comparative and Experimental Medicine Program, University of Tennessee Veterinary Teaching Hospital, Knoxville, United States 2 Department of Comparative Medicine, University of Tennessee Veterinary Teaching Hospital, Knoxville, United States 3 Department of Pathobiology, University of Tennessee Veterinary Teaching Hospital, Knoxville, United States Keywords: Viral IL-10; Malignant catarrhal fever; Ovine herpesvirus-2; IL-10homologue
Species: Ruminants Interleukin-10 (IL-10) interferes with monocyte and macrophage activation of Th1 helper lymphocyte production of nitric oxide and synthesis of various inflammatory mediators. An IL-10homologue (vIL-10) is produced by several herpes viruses and is hypothesized
to help the virus down regulate, and thus evade, host immune responses. The gammaherpes rhadinovirus Ovine herpesvirus-2 (OvHV-2) encodes an IL-10 like molecule highly homologous to mammalian IL-10. This virus causes sheep-associated malignant catarrhal fever (MCF), the most common form of MCF in the United States. MCF is a lymphoproliferative and inflammatory syndrome which has delayed clinical presentation hypothesized to be influenced by the production of vIL-10. For this study, the gene encoding vIL-10, Ov2.5 ORF, was amplified by PCR, cloned, sequenced, and a predicted 30 amino acid segment from the amino terminus synthesized. This synthetic peptide was used to develop a novel direct enzyme-linked immunosorbant assay (ELISA) to detect isotype-specific antibodies to OvHV-2 vIL-10. Work to date indicates that lambs do not have detectable levels of maternally derived antibody to vIL-10 during the first few weeks of age. Ewes, which are refractory to clinical infection, generally have high levels of antibody to vIL-10. A weak correlation exists between the vIL-10 ELISA and a commercially available competitive-inhibition ELISA (CI-ELISA). We believe the vIL-10 ELISA will refine the ability to identify ruminants exposed to sheep-associated MCF, provide an important tool for determining the role of vIL-10 in disease pathogenesis, and may contribute toward the development of a new vaccine strategy for the control of malignant catarrhal fever. doi:10.1016/j.vetimm.2008.10.053 Immunoproteomic analysis of the protective outer membrane fraction of Anaplasma marginale Wendy C. Brown Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, United States Keywords: Mass spectrometry; Anaplasma marginale; Outer membranes; T lymphocytes E-mail address: [email protected]. Species: Ruminants Rickettsial pathogens in the genera Anaplasma and Ehrlichia cause acute infection in immunologically naïve hosts and are major causes of tick-borne disease in animals and humans. Immunization with Anaplasma marginale purified outer membranes induces complete protection against anaplasmosis in 75% of cattle, whereas immunization with the well-studied and immunodominant major surface proteins such as MSP2 has provided little or no protection. The completed genome sequence of A. marginale facilitated the identification of subdominant and less abundant immunogenic proteins in the outer membrane fraction, using two approaches. First, two-dimensional electrophoresis and immunoblotting of the outer membrane fraction with immune bovine sera identified numerous antigenic protein spots. Analysis of individual proteins excised from the gels by liquid chromatography and tandem mass spectrometry identified 21 novel antigens. Of particular interest is the finding that three proteins from the type IV secretion system, conju-