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.

Chronic Inflammatory Disease in the Pancreas, Kidney and Salivary Glands of English Cocker Spaniels and Dogs of Other Breeds Shows Similar Histological Features to Human IgG4-related Disease.

Chronic pancreatitis (CP) is a common disease in the English cocker spaniel (ECS) and is characterized histologically by duct destruction, interlobular fibrosis and dense periductular and perivenous lymphocytic aggregates. These features are also found in human autoimmune pancreatitis type 1, part of a glucocorticoid-responsive, multiorgan syndrome, newly recognized as IgG4-related disease (IgG4-RD). Human IgG4-RD affects one or several organs, often showing a predominance of IgG4+ plasma cells histologically, with an IgG4+:total IgG+ plasma cell ratio of >40%. This study investigated whether ECSs with CP and/or inflammatory disease in several organs show an increase in IgG4+ plasma cells within affected tissues. Histological sections of pancreas, liver, kidney, salivary gland and conjunctiva were obtained from ECSs with idiopathic chronic inflammatory disease affecting those tissues. Tissue samples from age-matched dogs of other breeds with similar diseases were also sampled. Control diseased tissue samples, from dogs without a suspected immune-mediated disease, were included. A subset of ECSs and dogs of other breeds presented with disease in more than one organ. Immunohistochemistry was performed with primary reagents detecting total IgG and three of the four canine IgG subclasses (IgG2, IgG3 and IgG4). Normal sections of pancreas and liver showed an absence of labelled plasma cells of any subclass. Normal kidney and salivary gland sections showed the presence of a few labelled plasma cells (<10 plasma cells/high-power field). Fourteen tissue sections from 12 ECSs and seven sections from six dogs of other breeds showed elevated numbers of IgG4+ plasma cells and IgG4+:IgG+ ratios >40%. Individual dogs (ECSs and other breeds) showed marked increases in IgG4+ cells. There were no significant differences in the number of IgG4+ plasma cells between ECSs and dogs of other breeds for affected pancreas, liver, salivary glands and conjunctiva. Kidney sections had more IgG4+ cells, for both ECSs and dogs of other breeds, than did sections from other organs. Dogs of other breeds had significantly more IgG4+ plasma cells in affected kidneys than ECSs. In conclusion, several ECSs and dogs of other breeds fulfilled the histological criteria for the diagnosis of IgG4-RD, supporting the existence of a multiorgan immune-mediated disease in ECSs and some dogs of other breeds.

T Lymphocytes in Histiocytic Sarcomas of Flat-Coated Retriever Dogs.

Flat-Coated Retriever dogs are predisposed to the development of histiocytic sarcoma (HS), a poorly differentiated, highly malignant neoplasm. The authors have previously documented a significant lymphocytic infiltrate in such tumors. The objective of this study was to examine the presence and expression of regulatory T cells in HS tumor samples. Forty tumors were included in this study. All tumors were immunolabeled for CD3, CD79a, CD25, CD45RA, and FOXP3. The proportion of positive cells was compared between tumors presenting as a localized primary soft tissue mass (soft tissue origin HS) and disseminated HS affecting viscera, especially the spleen (splenic origin HS). By immunohistochemistry, 95% of infiltrating T cells were positive for Foxp3 in all sections, suggesting the presence of regulatory T cells. The proportion of cells positive for FOXP3 was higher in the tumors arising in soft tissues, whereas the proportion of CD45RA-positive cells was higher in the splenic origin HS. Canine HS has an aggressive clinical behavior and is uniformly fatal. The difference in the proportion of tumor-infiltrating lymphocytes positive for these 2 markers in the 2 locations may represent differences in tumor microenvironment between the 2 sites.

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.

Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes.

To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-gamma gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.

Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes.

Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)-DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-gamma in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.

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.

Recombinant single-chain Fv antibodies that recognize the p25 protein of the Maedi-Visna virus.

Single chain Fv (scFv) antibodies (generated by phage display technology, molecules representing new and efficient tools in the research and diagnostics of infectious diseases) against the capsid protein (p25) of Maedi-Visna virus were selected. Several clones of p25 specific scFv antibodies were identified; one of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and further characterized by sequencing and determination of the kinetic equilibrium association constant. Sequence analysis showed that the rearranged VL and VH domains of the analyzed scFv clone used sequences from the VL3 family (germline DPL16/VL3.1) and VH1 family (germline VH20), respectively. The kinetic equilibrium association constant was determined as KA = 1.12 +/- 0.52 L/mumol.

Infection of dendritic cells by the Maedi-Visna lentivirus.

The early stages of lentivirus infection of dendritic cells have been studied in an in vivo model. Maedi-visna virus (MVV) is a natural pathogen of sheep with a tropism for macrophages, but the infection of dendritic cells has not been proven, largely because of the difficulties of definitively distinguishing the two cell types. Afferent lymphatic dendritic cells from sheep have been phenotypically characterized and separated from macrophages. Dendritic cells purified from experimentally infected sheep have been demonstrated not only to carry infectious MVV but also to be hosts of the virus themselves. The results of the in vivo infection experiments are supported by infections of purified afferent lymph dendritic cells in vitro, in which late reverse transcriptase products are demonstrated by PCR. The significance of the infection of afferent lymph dendritic cells is discussed in relation to the initial spread of lentivirus infection and the requirement for CD4 T cells.

In vivo depletion of CD8+ cells does not affect primary maedi visna virus infection in sheep.

T-cells have been implicated both, in promoting and reducing viral replication during lentivirus infection. CD8+ lymphocytes are believed to be important in controlling viral load through direct killing of virus-infected cells and by secretion of inhibitory chemokines and cytokines. To evaluate the role of CD8+ T-cells in the induction and control of the primary phase of a lentivirus infection, we have used a non-T-cell tropic lentivirus, maedi-visna virus (MVV), to study the initial pathogenesis and subsequent immune responses in sheep depleted in vivo of CD8+ cells. Sheep were depleted of CD8+ cells in both blood and efferent lymph for up to 14 days. No difference in MVV replication was observed in either the draining efferent lymph or lymph node of these sheep. Surprisingly, these animals displayed a normal induction of pCTL whereas the virus-specific proliferative responses were reduced. This could reflect either that a proportion of functional CD8+ lymphocytes remained in these animals, as suggested by the appearance of pCTLs, or that CD8+ cells are not required for control of primary MVV infection.

CD4(+) T-cells are required for the establishment of maedi-visna virus infection in macrophages but not dendritic cells in vivo.

The role of CD4(+) lymphocytes in the establishment of lentivirus infection in macrophages has been studied in an in vivo system of lentivirus infection where CD4(+) lymphocytes are not the targets for infection. Using the non-T-cell-tropic lentivirus, maedi-visna virus (MVV), in CD4-depleted sheep, we have found that CD4(+) T cells were required for MVV infection in macrophages but not dendritic cells. CD4-depleted sheep had significantly lower levels of MVV-infected cells in lymph nodes and efferent lymph after MVV challenge in the drainage area of the lymph node. Due to the absence of virus in combination with the lack of CD4(+) T helper cells, virus-specific immune responses were reduced. There was delayed induction of cytotoxic T cell precursors, a marked reduction in virus-specific in vitro proliferative responses, and a delay in the appearance of MVV-specific antibodies. By contrast, CD4 depletion had no effect on the establishment of MVV infection in afferent lymph dendritic cells migrating from the skin infection site to the lymph node.