320 ESVP/ECVP Proceedings 2010 143:4, 2010

O3BRAIN INFECTION FOLLOWING EXPERIMENTAL

STAPHYLOCOCCUS AUREUS SEPSIS IN PIGSL.B. Astrup, T. Iburg, O.L. Nielsen,

H.E. Jensen, P.S. Leifsson and J.S. AgerholmUniversity of Copenhagen, Denmark

Introduction: Sepsis is a major problem in man and the associatedincidence and mortality is increasing. Multiple microabscesses canbe found in the brain of septic patients. Staphylococcus aureus is one ofthe most common causes of sepsis and brain abscesses. S. aureus isalso a frequent cause of spontaneous porcine pyaemia, including en-docarditis, and associated brain lesions. We present a porcine modelof haematogenous S. aureus-induced brain infection.Materials and Methods: Twelve pigs received an intravenous in-jection of S. aureus of 108 CFU/kg body weight once at 0 h or twiceat 0 h and 12 h. Four pigs were kept as controls. The pigs were killedin groups of four at 6, 12, 24 or 48 h post-infection. The brain was col-lected from all the animals and examined histologically.Results: All of the inoculated pigs developed sepsis and seven of 12animals had microabscesses in the prosencephalon. The brain lesionsshowed a temporal progression.Conclusions: The brain lesions in the experimentally infected pigswere similar to the brain lesions observed in pigs with spontaneous en-docarditis and those observed in human patients with septic enceph-alopathy. These findings validate this porcine model for futureresearch.

O4ANAPLASMA PHAGOCYTOPHILUM e SITES OF

PERSISTENT INFECTION IN SHEEPJ. Williams, Z. Woldehiwet, U. Hetzel and A. Kipar

Liverpool University, UKIntroduction: Anaplasma phagocytophilum (AP) is an intracellular rick-ettsial organism and is the causative agent of tick borne fever (TBF)in ruminants. AP is transmitted by the tick Ixodes ricinus and infectioncauses pyrexia, thrombocytopenia and immune suppression. After in-fection, the organism causes bacteraemia followed by prolonged per-sistence in tissues with possible subsequent bouts of bacteraemia. Inacute infection the organism has been demonstrated in alveolar mac-rophages, Kupffer cells and other tissue macrophages. Sites of persis-tence have yet to be established.Materials and Methods: Twelve na€ıve cross-bred lambs were in-fected intravenously with a TBF variant of AP and surveyed for aninterval between three weeks and 12 months. A range of tissues wasassessed by light microscopy, immunohistochemistry, DNA in-situ hy-bridization, transmission electron microscopy and quantitative PCRfor AP DNA.Results: Lung and liver were identified as sites of persistent infectionin experimentally infected sheep. Pulmonary intravascular macro-phages and hepatic Kupffer cells were shown to carry the agent.Conclusions: AP persists in specific tissue macrophages in constantcontact with the blood. These have been shown to also be the primarysite of AP infection prior to bacteraemia.

O5OF MAN AND PIGS: NEW INSIGHTS INTO THE

PATHOGENESIS OF CLOSTRIDIUM PERFRINGENS TYPEC ENTERITIS

F. Popescu*, C. Gurtner*, M. Wyder*, D. Authemann y

and H. Posthaus**Institute of Animal Pathology, Vetsuisse Faculty and yInstitute of Infectious

Diseases, Faculty of Medicine, University of Bern, SwitzerlandIntroduction: Clostridium perfringens type C causes fatal necrotizingenteritis in animals and man. The main virulence factor of pathogenicstrains is beta-toxin (CPB), a member of the beta-barrel pore-formingtoxin family. Previous immunohistochemical studies have shown CPBbinding in thrombotic vessels in intestinal lesions. To investigatewhether endothelial cells are specifically targeted by beta toxin, weestablished a novel in-vitro system using primary porcine and humanendothelial cells and recombinant CPB (rCPB).Materials and Methods: Primary porcine and human endothelialcells were incubated with different concentrations of clostridial cul-ture supernatants and rCPB expressed in E. coli. Cellular effectswere monitored using live cell imaging, immunofluorescence, cell vi-ability assays, FACS analyses and western blot analyses of membranefractions.Results: Both culture supernatants and rCPB induced marked cyto-pathic and cytotoxic effects, with a rapid disruption of the actin cyto-skeleton, cell border retraction and cell shrinkage followed by celldeath. These effects led to disintegration of the cell monolayer andwere neutralized with monoclonal antibodies against CPB.Conclusions: CPB may mediate the disruption of the endothelialbarrier leading to vascular thrombosis and ischaemic tissue necrosisin Clostridium perfringens type C enteritis.

O6THE ROLE OF ANTIGEN PRESENTING CELLS ANDT LYMPHOCYTES IN THE TONSIL OF PORCINEREPRODUCTIVE AND RESPIRATORY SYNDROME

VIRUS-INFECTED PIGSI.M. Rodriguez-Gomez*, J. Gomez-Laguna*, I. Barranco*,F.J. Salguero y, F.J. Pallares z, L. Carrasco* and G. Ramisz*Department of Anatomy and Comparative Pathology, Faculty of VeterinaryMedicine, Cordoba University, Spain, yVeterinary Laboratories Agency, UKand zDepartment of Anatomy and Comparative Pathology, Faculty of Veterinary

Medicine, Murcia University, SpainIntroduction: Porcine reproductive and respiratory syndrome(PRRS) is an economically important disease in swine production.PRRS virus modulates the host immune response, but the mecha-nisms involved remain unclear. The aim of this study was to evaluatechanges in the subpopulations of antigen presenting cells and T lym-phocytes in the tonsils of PRRS virus-infected pigs.Materials and Methods: Twenty-eight piglets were randomly dis-tributed into groups of four and killed at 3, 7, 10, 14, 17, 21 and 24days post-inoculation (dpi). Four identical pigs were used as a controlgroup and killed at the end of the study. Tonsil samples were collectedand fixed in 10 % neutral buffered neutral formalin and in Bouin’s so-lution. Antibodies against PRRS virus, SWC3, S-100, HLA-DR andCD3 antigens were used in an immunohistochemical study.Results: Viral antigenwasmainly expressed in the cytoplasmofmacro-phages and less commonly in interdigitating dendritic cells, with a peakat 7 dpi. The different cell subpopulations showed undulating kinetics,with a mild increase in macrophages and follicular dendritic cell countsat 7 and 10 dpi, respectively, and a decrease in the expression of majorhistocompatibility complex molecules and T lymphocytes at 3 dpi.Conclusions: PRRS virus may cause ineffective activation of anti-gen presenting cells and T lymphocytes activation. This study wassupported financially by the Spanish Ministry of Science and Innova-tion (AGL2009-12438).