Dual role of splenic mononuclear and polymorphonuclear cells in the outcome of ciprofloxacin treatment of Salmonella enterica infections.

OBJECTIVES: To determine the immune cell populations associated with Salmonella enterica serovar Typhimurium before and after ciprofloxacin treatment using a murine model of systemic infection. The effect of depletion of immune cells associating with Salmonella on treatment outcome was also determined. METHODS: We infected mice with a Salmonella enterica serovar Typhimurium strain expressing GFP and used multicolour flow cytometry to identify splenic immune cell populations associating with GFP-positive Salmonella before and after treatment with ciprofloxacin. This was followed by depletion of different immune cell populations using antibodies and liposomes. RESULTS: Our results identified CD11b+CD11chi/lo (dendritic cells/macrophages) and Ly6G+CD11b+ (neutrophils) leucocytes as the main host cell populations that are associated with Salmonella after ciprofloxacin treatment. We therefore proceeded to test the effects of depletion of such populations during treatment. We show that depletion of Ly6G+CD11b+ populations resulted in an increase in the number of viable bacterial cells in the spleen at the end of ciprofloxacin treatment. Conversely, treatment with clodronate liposomes during antimicrobial treatment, which depleted the CD11b+CD11chi/lo populations, resulted in lower numbers of viable bacteria in the tissues. CONCLUSIONS: Our study identified host cells where Salmonella bacteria persist during ciprofloxacin treatment and revealed a dual and opposing effect of removal of Ly6G+CD11b+ and CD11b+CD11chi/lo host cells on the efficacy of antimicrobial treatment. This suggests a dichotomy in the role of these populations in clearance/persistence of Salmonella during antimicrobial treatment.

Clinical and histopathological characterization of a large animal (ovine) model of collagen-induced arthritis.

Collagen induced arthritis (CIA) is the most studied and used rheumatoid arthritis (RA) model in animals, as it shares many pathological and immunological features of the human disease. The aim of this study was to characterize clinical and immunological aspects of the ovine CIA model, and develop lameness and histopathological scoring systems, in order to validate this model for use in therapeutic trials. Sheep were sensitized to bovine type II collagen (BCII), arthritis was induced by injection of bovine collagen type II into the hock joint and the response was followed for two weeks. Clinical signs of lameness and swelling were evident in all sheep and gross thickening of the synovium surrounding the tibiotarsal joint and erosion on the cartilage surface in the arthritic joints. Leucocyte cell counts were increased in synovial fluid and there was synovial hyperplasia, thickening of the intimal layer, inflammation and marked angiogenesis in the synovial tissue. There was a large influx of monocytes and lymphocytes into the synovial tissue, and increased expression of TNF-α and IL-1ß in arthritic intima, angiogenesis and upregulation of VCAM-1. CIA in sheep appears to be an excellent large animal model of RA and has the potential for testing biological therapeutics for the treatment of rheumatoid arthritis.

Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

Use of B7 costimulatory molecules as adjuvants in a prime-boost vaccination against Visna/Maedi ovine lentivirus.

RNA transcripts of the B7 family molecule (CD80) are diminished in blood leukocytes from animals clinically affected with Visna/Maedi virus (VMV) infection. This work investigates whether the use of B7 genes enhances immune responses and protection in immunization-challenge approaches. Sheep were primed by particle-mediated epidermal bombardment with VMV gag and env gene recombinant plasmids together with plasmids encoding both CD80 and CD86 or CD80 alone, boosted with gag and env gene recombinant modified vaccinia Ankara virus and challenged intratracheally with VMV. Immunization in the presence of one or both of the B7 genes resulted in CD4+ T cell activation and antibody production (before and after challenge, respectively), but only immunization with CD80 and CD86 genes together, and not CD80 alone, resulted in a reduced number of infected animals and increased early transient cytotoxic T lymphocytes (CTL) responses. Post-mortem analysis showed an immune activation of lymphoid tissue in challenge-target organs in those animals that had received B7 genes compared to unvaccinated animals. Thus, the inclusion of B7 genes helped to enhance early cellular responses and protection (diminished proportion of infected animals) against VMV infection.

CCH cells are potent stimulators in the allogeneic mixed leucocyte reaction.

Canine cutaneous histiocytoma (CCH) has been identified as a tumour of epidermal Langerhans cells (LCs) on the basis of immunophenotypic studies. Neoplastic Langerhans cells (CCH-LCs) were isolated from lesions of canine cutaneous histiocytoma. The CCH-LC cells expressed CD1b, CD11/18, CD45, MHC-I, and MHC-II. The CCH-LC cells were potent stimulators of the mixed leucocyte reaction (MLR) in vitro when compared to PBMCs from the tumour-bearing animals. This provides evidence that the neoplastic cells in CCH have functional as well as immunophenotypic characteristics of Langerhans cells.

Cloning and sequencing of ovine Flt3 ligand.

A cDNA (879 bp) containing the complete open reading frame of ovine Fms-related tyrosine kinase 3 ligand (Flt3-L) was amplified by reverse transcriptase polymerase chain reaction (RT-PCR), cloned and sequenced. The deduced amino acid sequence has 97.6% similarity with bovine Flt3-L isoform 1 and shares the long cytoplasmic domain observed in bovine Flt3-L but not in human Flt3-L.

H2-M3 major histocompatibility complex class Ib-restricted CD8 T cells induced by Salmonella enterica serovar Typhimurium infection recognize proteins released by Salmonella serovar Typhimurium.

Salmonella enterica serovar Typhimurium causes a typhoid-like disease in mice which has been studied extensively as a model for typhoid fever in humans. CD8 T cells contribute to protection against S. enterica serovar Typhimurium in mice, but little is known about the specificity and major histocompatibility complex (MHC) restriction of the response. We report here that CD8 T-cell lines derived from S. enterica serovar Typhimurium-infected BALB/c mice lysed bone marrow macrophages infected with S. enterica serovar Typhimurium or pulsed with proteins from S. enterica serovar Typhimurium culture supernatants. Cytoxicity was beta-2-microglobulin dependent and largely TAP dependent, although not MHC class Ia restricted, as target cells of several different MHC haplotypes were lysed. The data suggested the participation of class Ib MHC molecules although no evidence for the presence of Qa1-restricted T cells could be found, unlike in previous reports. Instead, the T-cell lines lysed H2-M3-transfected fibroblasts infected with S. enterica serovar Typhimurium SL3261 or treated with Salmonella culture supernatants. Thus, this report increases the number of MHC class Ib antigen-presenting molecules known for Salmonella antigens to three: Qa-1, HLA-E, and now H2-M3. It also expands the range of pathogens that induce H2-M3-restricted CD8 T cells to include an example of gram-negative bacteria.

Neutrophilic dermatitis and immune-mediated haematological disorders in a dog: suspected adverse reaction to carprofen.

This report describes the clinical and pathological findings of a suspected idiosyncratic adverse drug reaction in a young dog. The patient presented with sudden onset, severe skin lesions together with episodes of collapse. Investigations revealed a neutrophilic dermatitis with vasculitis, immune-mediated haemolytic anaemia and thrombocytopenia. Similar pathology has been described in human cases of Sweet's syndrome. The chronology of events suggested an adverse drug reaction to carprofen, although two antibiotics had been prescribed within the dog's recent history. Lymphocyte transformation tests were performed and tended to exclude both antibiotics as the cause of the reaction. To the authors' knowledge, lymphocyte transformation tests have not previously been described with regard to drug hypersensitivity assessment in the veterinary literature, and this is the first peer-reviewed case report of neutrophilic dermatitis and vasculitis with immune-mediated haemolytic anaemia and thrombocytopenia occurring as a suspected adverse drug reaction to carprofen in the dog.

Diagnostic tests for small ruminant lentiviruses.

Maedi visna virus and caprine arthritis encephalitis virus are closely related retroviruses that cause chronic inflammatory disease in small ruminants. The infections are characterised by insidious onset and slow progression. Diagnosis of infection is usually by serological testing. A variety of assays are available for this purpose, though the relative sensitivity and specificity of these assays has not been compared systematically. Here we review recent developments in laboratory diagnostic methods and their use in field diagnosis. The results suggest that a combination of ELISA and PCR might afford optimal detection of SRLV infection.

Transmission of small ruminant lentiviruses.

Small ruminant lentiviruses (SRLV) are classical slow retroviruses causing chronic inflammatory disease in a variety of target organs. The routes of transmission have been investigated and a large body of evidence has accumulated over many years. The main routes are through ingestion of infected colostrum and/or milk, or through inhalation of respiratory secretions. However, many studies also provide evidence that intrauterine infection may occur, though the extent and significance of this route is controversial. Embryos treated to IETS standards appear to pose very little risk of infection. SRLV have been detected in semen suggesting a potential source of transmission. However, such transmission has not been demonstrated to date. The application of control measures based on this information allows more efficient strategies to be developed which will reduce the rate of transmission.

The phenotype and phagocytic activity of macrophages during maedi-visna virus infection.

Macrophages from maedi-visna virus (MVV) infected sheep have been shown to have an activated phenotype from sites of lesions in vivo. Here we have looked at the direct effect of virus infection on macrophage phenotype and activity in vitro by flow cytometry. There was no significant difference in the expression of several surface markers (CD4, CD8, MHC Class I, MHC Class II, lymphocyte function associated antigen(LFA)-1 and LFA-3) on monocyte-derived macrophages (MDM) by 5 days post MVV infection. In contrast the phagocytic activity of MVV-infected MDM for the yeast Candida utilis and erythrocytes was decreased by 5 days p.i. although the surface binding of erythrocytes was not affected. Interestingly, an activated phenotype was seen on alveolar macrophages (AM) from sheep with maedi (surface expression of MHC Class I, Class II and LFA-1 was increased), but there was no difference in the binding and phagocytosis of erythrocytes by these cells. However the binding and phagocytosis of the bacterium, Pasteurella hemolytica was increased with AM from MVV-infected sheep without lesions. Similarly there was no significant difference in the phagocytic and erythrocyte rosetting activity between fresh monocytes from MVV-infected and uninfected control sheep. Therefore the phenotype of macrophages taken from sites of lesions caused by MVV does not correspond to a direct effect by the virus on these cells or to particular activities of the macrophages.

Electron microscope studies of the replication of a British isolate of maedi visna virus in macrophages and skin cell lines.

The replication of EV1, a British isolate of maedi visna virus (MVV), in macrophages has not previously been studied. We therefore used transmission and scanning electron microscopy (TEM and SEM respectively) to compare the replication of EV1 in macrophages versus skin cell lines. Monocyte-derived macrophages (MDM), alveolar macrophages (AM) and skin cell lines were all permissive for replication by EV1. Virus grew rapidly and to high titers in skin cell lines and mature MDM. However replication was slower in less mature MDM or AM. Virion budding occurred through (i) cytoplasmic membranes only (skin cells), (ii) cytoplasmic and vesicular membranes (MDM) or (iii) vesicular membranes only (AM). This meant that virions accumulated in vacuoles within macrophages. Retroviral intracytoplasmic type A particles were seen in the cytoplasm of AM and MDM infected with strain EV1, but not MDM infected with strain 1514 (an Icelandic MVV strain) and were shown to contain MVV gag antigen by immunogold staining.

The restricted IgG1 antibody response to maedi visna virus is seen following infection but not following immunization with recombinant gag protein.

Maedi-visna (MVV) is a retrovirus of the subfamily lentivirinae which includes HIV, simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Infection of its natural host, the sheep, does not cause overt immunodeficiency, but rather a chronic inflammatory disease. However, subtle immunological changes following infection have been reported including a sheep IgG1 subclass-restricted MVV-neutralizing antibody. Here we demonstrate by Western blotting that there is no IgG2 serum antibody response to any MVV antigen after MVV infection, in contrast to infection with the parapox virus Orf, when serum IgG2 anti-Orf antibody is readily detected. By ELISA, the IgG1 antibody titres to Orf are higher than to MVV, but the minimum MVV serum antibody IgG1/IgG2 ratio is significantly raised compared with that for Orf virus antibody in the same sheep, indicating that the IgG2 defect in MVV infection cannot be accounted for by differences in the sensitivity of the Orf and MVV ELISA. Serum IgG2 anti-MVV gag p. 25 can be detected in both normal and MVV-infected sheep following immunization with purified recombinant MVV gag p 25 protein in Freund's complete adjuvant. The failure to make an IgG2 MVV-specific antibody indicates that immunological dysfunction can arise with macrophage tropic lentiviruses, and it may aid viral persistence.

Initial lentivirus-host interactions within lymph nodes: a study of maedi-visna virus infection in sheep.

Reactive changes occurring within lymph nodes draining the subcutaneous site of acute infection with maedi-visna virus (MVV) were studied, and the appearance of infected cells correlated with the immune response. Cells infected with virus were detected in the node by cocultivation from day 4 postinfection (p.i.), with maximum numbers being seen between days 7 and 14, but even then infected cells were rare, with a maximum frequency of 23 50% tissue culture infective doses (TCID50) in 10(6) lymph node cells. At later times, infected cells were still detected, but their numbers fell to 1 to 2 TCID50 per 10(6) cells. Virus-specific CD8+ cytotoxic T-cell precursors (CTLp) were isolated from infected nodes from day 10 p.i. onwards, and T-cell proliferative responses to MVV were first detected on day 7 and consistently detected after day 18. Histological analysis showed a vigorous immune response in the node. There was a marked blast reaction in the T-cell-rich zones, which was greatest at the time when the number of virally infected cells was at its height. At this stage, large numbers of plasma cells were seen in the medullary cords, indicating that extensive T-cell-dependent B-cell activation was occurring in the T-cell-rich zones. Germinal centers were prominent shortly after the onset of the T-zone response and were still present at 40 days p.i. Phenotype studies of isolated lymph node cells failed to detect major changes in the proportion or phenotype of macrophages, CD1+ interdigitating cells, and CD4+ or CD8+ T cells despite the fact that CD8+ lymphoblasts form a major population leaving the node in efferent lymph. This suggests that there is a balanced increase in the number of all cell types in response to the virus within the node and selective migration of CD8+ lymphoblasts containing virus-specific CTLp from the node. Virus-specific immune responses are therefore present within the node when infectious virus isolation is maximal, but cellular immunity may act to control the level of infection from day 18 onwards.

Cytotoxic activity against maedi-visna virus-infected macrophages.

The cell type predominantly infected by maedi-visna virus (MVV) is the macrophage, and we have looked at the ability of MVV-infected macrophages to interact with cytotoxic T lymphocytes (CTL), important effector cells against virus infections. MVV-specific CTL precursors were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in peripheral blood lymphocytes of all MVV-infected sheep. MVV-infected monocyte-derived macrophages and alveolar macrophages were able to stimulate CTL activity in vitro and were targets for these activated CTL. The major effector cell population using MVV-infected macrophage targets was CD8+ lymphocytes, although another population, lymphokine-activated killer cells, may also have been involved. There was no direct cytotoxic activity found in alveolar lymphocytes from MVV-infected sheep without lung lesions.

Circulating cytotoxic T lymphocyte precursors in maedi-visna virus-infected sheep.

Maedi-visna virus (MVV)-specific cytotoxic T lymphocytes (CTL) were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in the efferent lymph and peripheral blood of sheep chronically infected with MVV. Cytotoxicity was mediated by CD8+ lymphocytes and was specific for particular strains of MVV. These precursor CTL were detected in the blood between day 23 and day 100 after infection via the skin. In one out of seven persistently infected sheep MVV-specific cytotoxicity was seen in uncultured peripheral blood cells. Again the effector population consisted of CD8+ lymphocytes. The only other viral infections in which CTL have been detected in peripheral blood mononuclear cells prior to secondary stimulation are those caused by the simian and human immunodeficiency viruses.

Characteristics of the T cell-mediated immune response to maedi-visna virus.

Virus-specific T cell-mediated immunity was investigated in healthy sheep persistently infected with the ruminant lentivirus maedi-visna. Visna-specific lymphocyte proliferation was demonstrated in response to both purified virions and recombinant p25, the major core protein of maedi-visna virus. The responding T cell population in this assay was mainly of the CD4+ phenotype, although in some individuals CD8+ T cells were also shown to respond to visna antigen in this system.