Efficient isolation of avian bornaviruses (ABV) from naturally infected psittacine birds and identification of a new ABV genotype from a salmon-crested cockatoo (Cacatua moluccensis).

Avian bornaviruses (ABV) have been discovered in 2008 as the causative agent of proventricular dilatation disease (PDD) in psittacine birds. To date, six ABV genotypes have been described in psittacines. Furthermore, two additional but genetically different ABV genotypes were recognized in non-psittacine birds such as canary birds and wild waterfowl. This remarkable genetic diversity poses a considerable challenge to ABV diagnosis, since polymerase chain reaction (PCR) assays may fail to detect distantly related or as yet unknown genotypes. In this study we investigated the use of virus isolation in cell culture as a strategy for improving ABV diagnosis. We found that the quail fibroblast cell line CEC-32 allows very efficient isolation of ABV from psittacine birds. Isolation of ABV was successful not only from organ samples but also from cloacal and pharyngeal swabs and blood samples collected intra vitam from naturally infected parrots. Importantly, using this experimental approach we managed to isolate a new ABV genotype, termed ABV-7, from a salmon-crested cockatoo (Cacatua moluccensis). Phylogenetic analysis showed that ABV-7 is most closely related to the psittacine genotypes ABV-1, -2, -3, and -4 and clearly distinct from genotypes ABV-5 and -6. Our successful identification of ABV-7 emphasizes the necessity to consider the high genetic diversity when trying to diagnose ABV infections with high reliability and further shows that classical virus isolation may represent a useful diagnostic option, particularly for the detection of new ABV genotypes.

Defining cytochemical markers for different cell types in the equine retina.

The major cell types in the mammalian retina are photoreceptors, amacrine, horizontal, bipolar, ganglion and Mueller glial cells. Most of the specific cell types are conserved, but cytochemical markers vary between species. The aim of our study was to characterize cytochemically distinctive markers for different cell types in the equine retina. We were able to define specific markers for equine Mueller glial cells and photoreceptor cells. Furthermore, we describe markers for large ganglion cells, horizontal and amacrine cells and a subpopulation of bipolar cells. Additionally, discrimination between the inner plexiform layer and nerve fibre layer can be achieved by expression of syntaxin and neurofilament 200 respectively.

An enzyme-linked immunosorbent assay (ELISA) for detection of Marek's disease virus-specific antibodies and its application in an experimental vaccine trial.

An enzyme-linked immunosorbent assay (ELISA) for the detection of Marek's disease virus (MDV)-specific antibodies was developed. Chicken embryo cells (CEC) or chicken kidney cells (CKC) were infected with MDV vaccine strain CVI988/Rispens, and infected-cell lysates were prepared at day 5 post-infection by freeze-thawing. Uninfected-cell lysates served as negative controls. Sera were used at a 1 : 100 dilution and were added in parallel to wells containing the infected and uninfected cell lysates. The optical densities at 492 nm (OD(492 nm)) were measured after detection of bound chicken antibodies with anti-chicken IgG peroxidase conjugate and colour reactions using o-phenylenediamine (OPD) as a substrate. The best results concerning the signal-to-noise ratio were obtained by using CKC cells rather than CEC for antigen preparation. The OD(492 nm) of plasma or serum samples with infected CKC was <0.02 when samples of unvaccinated and unchallenged maternal antibody-negative white leghorn chickens were tested. Sera and plasma samples of positive control birds exhibited OD(492 nm) of <0.01 when tested with uninfected CKC. The assay was used to monitor a trial that compared experimental BAC DNA vaccines and a commercial vaccine. Sustained seroconversion and antibody titers that were constantly rising until day 84 after vaccination (71 days after challenge) was observed only when chickens did not develop Marek's disease. In contrast, chickens developing the disease mounted marginal and short-lived antibody titers only. We conclude that the developed ELISA may be a valuable tool for the evaluation of the efficacy of MDV vaccination under experimental but possibly also under field conditions.

[Clinical, hematological, biochemical and endocrinological aspects of 32 dogs with hypothyroidism]. / Klinische, hämatologische, biochemische und endokrinologische Aspekte bei 32 Hunden mit Hypothyreose.

During the years of 1996-2001, hypothyroidism was diagnosed at the clinic for small animal internal medicine, University of Zurich, in 32 dogs. Most of the dogs were large breeds. The most frequent clinical characteristics observed were exercise intolerance, obesity, dermatological, neurological and gastrointestinal signs. Predominant laboratory abnormalities were a low red blood cell count, increased concentration of cholesterol, triglycerides and fructosamin. 29 dogs had a T4 below the reference range (< 1.5 micrograms/dl), one dog had a T4 at the lower limit thereof (1.6 micrograms/dl). One dog had a T4 within the reference range (3.4 micrograms/dl), another had a very high T4 of 206.8 micrograms/dl; the results of the latter 2 dogs were interpreted as incorrectly increased T4 values due to in vitro interference with T4-autoantibodies. Diagnosis was confirmed in all of the dogs based on TSH-stimulation testing. Endogenous TSH (cTSH) measured parallelly, was elevated in only 60% of the dogs. In about 67% of the dogs, hypothyroidism was associated with thyroglobulin-autoantibodies. Canine hypothyroidism is a rather rare endocrine disorder in Switzerland. The TSH-stimulation test remains the gold standard in confirming the disease; a definitive diagnosis can be challenging for practitioners because bovine TSH, used for the TSH-stimulation test is not licensed for use in dogs. Since assessment of cTSH using current assays shows normal values in a high percentage of hypothyroid dogs, the diagnostic value is only limited. In most of the hypothyroid dogs T4 is decreased, with the presence of autoantibodies to T4, it can be normal or increased.

Immunopathology of recurrent uveitis in spontaneously diseased horses.

Equine recurrent uveitis (ERU) is the most serious eye disease in horses worldwide. Despite the fact that ERU is generally considered to be immune mediated, a detailed description of the histopathology of the posterior part of ERU eyes is lacking. Here, we examined sections of paraffin-embedded eyes using histological and immunhistological methods. Twenty seven eyes of 20 horses with ERU and 30 eyes of 15 healthy control horses were included in this study. We could consistently demonstrate an involvement of the retina and the choroid in all examined eyes of horses with spontaneous ERU. In eyes with minimal histopathological changes, the infiltrates consisted almost exclusively of T-cells. Histopathological changes start with the destruction of the photoreceptor outer segments, which often leads to focal retinal detachment. In more severely affected eyes, there is additional disintegration of the ganglion cell layer and the inner nuclear layer. In almost all examined eyes, lymphoid follicle formation could be demonstrated. Typical localizations of these follicles were the iris stroma and the choroid underneath the transition zone of the retina without photoreceptor cells to the region containing photoreceptor cells. These follicles consist of a T-cell rich periphery with a small center of CD3-negative lymphocytes. In cases with extreme histopathological changes, the retinal architecture is widely disintegrated with massive infiltration of the retina, the choroid, and the ciliary body by several types of inflammatory cells. Necrotic remnants of the retina are end-stage findings and there is only a minor inflammatory infiltration left. This study provides clear evidence that the retina is involved in all stages of ERU. Inflammation is mainly driven by T-cells as T-cells were demonstrated in mild stages of the disease and are also the predominating cell type in all other stages of ERU.

Diverse expression of the K-1 antigen by cortico-medullary and reticular epithelial cells of the bursa of Fabricius in chicken and guinea fowl.

The immunocytochemical study of the K-1 monoclonal antibody indicates that the epithelial components of the bursa of Fabricius of the chicken and guinea fowl express the K-1 positive molecule. During embryogenesis, the K-1 antigen expression appears together with the bud-formation. As the number of B cells increases in the developing follicle, the K-1 expression gradually diminishes in the medullary reticular epithelial cells and completely ceases by hatching, which suggests that the molecule is developmentally regulated. After hatching, the expression of the molecule is restricted to the sealing off zone of the lymphoepithelial or medullary region of the follicle: i.e. to the cortico-medullary (CM) epithelial cells and the follicle associated epithelium (FAE) supporting cells in guinea fowl and to the latter ones in the chicken. The expression of the K-1 antigen by these epithelial components may support their structural identity. After hatching, the K-1 molecule is restricted to the CM epithelial cells and/or FAE supporting cells, which suggests that the function of the embryonic epithelial bud is taken over by the CM epithelial cells. The K-1 positive CM epithelial cells form arches, which encompass blast-like cells. The possible relationship of the CM epithelial cells and blast-like cells, which may represent the precursors of bursal secretory dendritic cells is discussed.

Chicken interleukin-6. cDNA structure and biological properties.

Suppression subtractive hybridization technology was used to identify differentially expressed genes in spleens of chickens that had been treated with the synthetic immune modifier S-28463. One induced chicken gene encoded a protein with about 35% sequence identity to human interleukin-6 (IL-6). It consists of 241 amino acids including a putative N-terminal signal peptide of 47 residues. Bacterially expressed chicken IL-6 (ChIL-6) carrying a histidine tag in place of the signal peptide was biologically active: it induced proliferation of the IL-6-dependent murine hybridoma cell line 7TD1. The concentration of ChIL-6 required for half-maximal proliferative response was approximately 60 pg.mL-1. When injected intravenously into adult chickens, purified recombinant ChIL-6 induced an increase in serum corticosterone levels. Supernatants of chicken LMH and monkey COS-7 cells transiently transfected with a ChIL-6 expression construct induced proliferation of 7TD1 cells, demonstrating that recombinant ChIL-6 from eukaryotic cells is also active.

NK and T cells constitute two major, functionally distinct intestinal epithelial lymphocyte subsets in the chicken.

Non-mammalian NK cells have not been characterized in detail; however, their analysis is essential for the understanding of the NK cell receptor phylogeny. As a first step towards defining chicken NK cells, several tissues were screened for the presence of NK cells, phenotypically defined as CD8(+) cells lacking T- or B-lineage specific markers. By this criteria, approximately 30% of CD8(+) intestinal intraepithelial lymphocytes (IEL), but <1% of splenocytes or peripheral blood lymphocytes were defined as NK cells. These CD8(+)CD3(-) IEL were used for the generation of the 28-4 mAb, immunoprecipitating a 35-kDa glycoprotein with a 28-kDa protein core. The CD3 and 28-4 mAb were used to separate IEL into CD3(+) IEL T cells and 28-4(+) cells, both co-expressing the CD8 antigen. During ontogeny, 28-4(+) cells were abundant in the IEL and in the embryonic spleen, where two subsets could be distinguished according to their CD8 and c-kit expression. Most importantly, 28-4(+) IEL lysed NK-sensitive targets, whereas intestinal T cells did not have any spontaneous cytolytic activity. These results define two major, phenotypically and functionally distinct IEL subpopulations, and imply an important role of NK cells in the mucosal immune system.

Modulation of functional activities of chicken heterophils by recombinant chicken IFN-gamma.

The objective of the present studies was to examine the in vitro effects of recombinant chicken interferon-gamma (rChIFN-gamma) on shape change, phagocytosis, and the oxidative/nonoxidative killing activities of day-old chicken heterophils. Heterophils (4 x 10(6)/ml) were incubated with various concentrations of recombinant ChIFN-gamma from both Escherichia coli and transfected Cos cells for 2 h at 39 degrees C. The incubation of the neonatal heterophils with rChIFN-gamma resulted in significantly greater numbers of cells with membrane shape change when compared with the mock-treated heterophils. Both Cos cell-derived and E. coli-derived ChIFN-gamma significantly increased (p < 0.01) the phagocytosis of opsonized or nonopsonized Salmonella enteritidis by the neonatal heterophils in a concentration-dependent manner. Incubation with ChIFN-gamma induced no direct stimulation of the respiratory burst by the chicken heterophils but did prime the heterophils for a significantly strengthened respiratory burst to subsequent stimulation with opsonized zymosan (OZ). Lastly, incubation of the heterophils with ChIFN-gamma primed the cells for a significant increase in the release of beta-D-glucuronidase following stimulation with OZ. These results show that neonatal avian heterophils can respond to cytokine modulation with enhanced functional competence, suggesting that ChIFN-gamma can enhance the immune competence of the innate defenses of chickens during the first week of life.

Immune responses to retinal autoantigens and peptides in equine recurrent uveitis.

PURPOSE: To test the hypothesis that autoimmune mechanisms are involved in horses in which equine recurrent uveitis (ERU) develops spontaneously. METHODS: Material obtained from horses treated for spontaneous disease by therapeutic routine vitrectomy was analyzed for total IgG content and IgG specific for S-Antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). The cellular infiltrate of the vitreous was analyzed by differential counts of cytospin preparations and flow cytometry using equine lymphocyte-specific antibodies. Antigen-specific proliferation assays were performed comparing peripheral blood lymphocytes (PBLs) with vitreal lymphocytes by stimulation with S-Ag and several S-Ag- and IRBP-derived peptides. RESULTS: The total IgG content of specimens from horses with ERU was very high with great variability among the investigated samples (11.5 +/- 8.0 mg). Autoantibodies to S-Ag or IRBP or both were found in 72% of vitreous specimens from horses with uveitis. The leukocyte infiltrates (up to 2 x 10(8) cells per sample) were dominated by lymphocytes (>90%) in most cases (22/32). Flow cytometry showed that more than 50% of these cells were CD4(+) T cells. In vitro stimulation of vitreal lymphocytes, but not of PBL, showed a strong proliferative response to peptides derived from S-Ag or IRBP in 9 of 12 patients. CONCLUSIONS: In the eyes of horses with ERU, IgG antibodies and autoreactive T cells specific for retinal antigens were detected. These results strongly support the hypothesis that ERU is an autoimmune-mediated disease and is highly similar to recurrent uveitis in humans in both clinical and immunologic parameters.

Cytokines of birds: conserved functions--a largely different look.

Targeted disruptions of the mouse genes for cytokines, cytokine receptors, or components of cytokine signaling cascades convincingly revealed the important roles of these molecules in immunologic processes. Cytokines are used at present as drugs to fight chronic microbial infections and cancer in humans, and they are being evaluated as immune response modifiers to improve vaccines. Until recently, only a few avian cytokines have been characterized, and potential applications thus have remained limited to mammals. Classic approaches to identify cytokine genes in birds proved difficult because sequence conservation is generally low. As new technology and high throughput sequencing became available, this situation changed quickly. We review here recent work that led to the identification of genes for the avian homologs of interferon-alpha/beta (IFN-alpha/beta) and IFN-gamma, various interleukins (IL), and several chemokines. From the initial data on the biochemical properties of these molecules, a picture is emerging that shows that avian and mammalian cytokines may perform similar tasks, although their primary structures in most cases are remarkably different.

Adjuvant effects of various lipopeptides and interferon-gamma on the humoral immune response of chickens.

The adjuvant effects of various lipopeptides and recombinant chicken interferon gamma (IFN-gamma) on the humoral immune response of laying hens was investigated in four immunization studies. We used the lipopeptide Pam3Cys-Ser-(Lys)4 (PCSL), the conjugate P-Th1 consisting of the lipopeptide P3CS and the T-helper epitope Th1 (FISEAIIHVLHSRHPG), and the conjugate P-Th2 of the lipopeptide P3CSS and the T-helper epitope Th2, which corresponds to the peptide EWEFVNTPPLV, as adjuvants. Human serum albumin (HSA), recombinant bovine somatotropin (RBST), and human immunoglobulin G (IgG) served as antigens in the different experiments. All tested adjuvants enhanced the humoral immune response with various intensities. Chickens showed high antibody titers after the immunization with HSA even without adjuvant, but the adjuvant effects of PCSL and the combination of PCSL and recombinant chicken interferon-gamma (IFN-gamma) were much more pronounced using the antigens RBST and IgG. Especially after the third immunization, higher titers of antibodies were induced by the coadministration of P-Th1 and, to a greater extent, by the combination of PCSL and P-Th1 compared with the use of PCSL. Also, chickens that had received PCSL and P-Th2 showed the highest immune response, even after the second booster. The average concentrations of chicken immunoglobulin Y were significantly higher in 5-mo-old chickens (9.4 mg/mL serum and 10.1 mg/mL egg yolk) compared with 9-mo-old chickens (5.9 mg/mL serum and 5.1 mg/mL egg yolk). The specific serum antibody response was higher in the older chickens than in the younger chickens. Because chicken antibodies are likely to be used increasingly for diagnostic and therapy in the future, lipopeptides and recombinant chicken IFN-gamma may find many applications as adjuvants, thus contributing to the welfare of experimental animals.

cDNA cloning of biologically active chicken interleukin-18.

By searching a chicken EST database, we identified a cDNA clone that appeared to contain the entire open reading frame (ORF) of chicken interleukin-18 (ChIL-18). The encoded protein consists of 198 amino acids and exhibits approximately 30% sequence identity to IL-18 of humans and various others mammals. Sequence comparisons reveals a putative caspase-1 cleavage site at aspartic acid 29 of the primary translation product, indicating that mature ChIL-18 might consist of 169 amino acids. Bacterially expressed ChIL-18 in which the N-terminal 29 amino acids of the putative precursor molecule were replaced by a histidine tag induced the synthesis of interferon-gamma (IFN-gamma) in cultured primary chicken spleen cells, indicating that the recombinant protein is biologically active.

Protective effects of type I and type II interferons toward Rous sarcoma virus-induced tumors in chickens.

Growth of tumors induced by Rous sarcoma virus (RSV) is controlled by alleles at the major histocompatibility complex locus in chickens, indicating that immunological host defense mechanisms play a major role. We show here that the resistance phenotype of CB regressor chickens can be partially reverted by treating the animals with a monoclonal antibody that neutralizes the major serotype of chicken type I interferon, ChIFN-alpha. Injection of recombinant ChIFN-alpha into susceptible CC progressor chickens resulted in a dose-dependent inhibition of RSV-induced tumor development. This treatment was not effective, however, in CC chickens challenged with a DNA construct expressing the v-src oncogene, suggesting that the beneficial effect of type I interferon in this system resulted from its intrinsic antiviral activity and probably not from indirect immunmodulatory effects. By contrast, recombinant chicken interferon-gamma strongly inhibited tumor growth when given to CC chickens that were challenged with the v-src oncogene, indicating that the two cytokines target different steps of tumor development.

Genes of chicken MHC regulate the adherence activity of blood monocytes in Rous sarcomas progressing and regressing lines.

The influence of the chicken major histocompatibility (B) complex (MHC) on the adherence potential of monocyte-derived macrophages was examined using the congenic chicken lines CB and CC. These lines represent well-defined genetic models for the study of resistance (CB) or susceptibility (CC) to the progressive growth of Rous sarcomas. Using a monoclonal antibody specific for chicken monocytes/macrophages, CB and CC chickens were shown by flow cytometry analyses to have similar proportions of peripheral blood monocytes. However, when the glass-adherence potential of these cells was compared during incubation in tissue culture medium over 24, 48 and 72 h at 40 degrees C, significant differences were seen between cells from these two inbred lines. After 24 and 48 h, glass-adherence by CB cells was 2-3 fold higher than that of CC cells. After 72 h this difference decreased to 1.5 fold. At 24 and 48 h, the adherent CB macrophages also appeared about 1.5 times larger than those of CC chickens. Genetic analysis using F1 hybrids (CBxCC) showed that this trait is regulated by a dominant gene that segregates with the B12 haplotype in the backcross generation F1xCC. From the results obtained with the recombinant congenic lines CB.R1 and CC.R1, we conclude that the gene regulating adherence potential is localized within the B-F/L region of the chicken MHC. About 50% of adherent cells were able to phagocytose opsonised FITC-labelled Zymosan particles. The level of nitric oxide production in vitro by CB and CC macrophages was equal. The importance of cells of the mononuclear phagocyte system for the response to Rous sarcoma virus (RSV) infection was studied in CB chickens using the anti-macrophage agents silica, carrageenan, and C12MDP, encapsulated in liposomes. In those chickens treated with silica and carrageenan, we observed progressive growth of RSV-induced tumors. The graft-versus-host reactivity of peripheral blood lymphocytes (PBL) of treated chickens was comparable to controls. In vitro nitric oxide production by macrophages from silica-treated chickens was higher than by macrophages from untreated controls.

Role of the humoral immune system in Salmonella enteritidis phage type four infection in chickens.

The role of avian humoral immunity in the clearance of S. enteritidis was evaluated through bursectomy. After oral inoculation of bursectomized and sham-treated chickens with S. enteritidis, faecal excretion of S. enteritidis was examined. Organs were collected weekly until six weeks post-inoculation (pi) for bacteriological enumeration. Antibody isotypes in serum and bile were quantified by ELISA. Faecal excretion of S. enteritidis was significantly lower in controls from 13 days pi. Numbers of S. enteritidis in caeca from controls were significantly decreased from three weeks pi. Numbers of S. enteritidis were significantly decreased at two weeks pi in the spleen and the liver and at six weeks pi in the liver. Antibodies to S. enteritidis peaked at two weeks pi in controls and were absent in bursectomized chickens. These findings indicate that elimination of S. enteritidis partly depends on humoral immunity. The intestinal humoral response appeared more effective than the systemic humoral response for elimination of S. enteritidis.

Inhibition of the development of spontaneous autoimmune thyroiditis in the obese strain (OS) chickens by in vivo treatment with anti-CD4 or anti-CD8 antibodies.

The involvement of CD4+ and CD8+ T cells in pathogenesis of spontaneous autoimmune thyroiditis (SAT) in obese strain (OS) chickens has not been studied in depth until now. We depleted CD4+ or CD8+ T cells in OS chickens by treatment with murine monoclonal anti-CD4 or anti-CD8 antibodies at 3 day intervals beginning at hatching. The birds were killed at 19-25 days of age. Treatment with anti-CD4 antibody completely prevented SAT development, while treatment with anti-CD8 antibody partially inhibited SAT. These results show the critical role of CD4+ T cells in the development of SAT in OS chickens, and indicate that CD8+ T cells are also involved in SAT pathogenesis.

Sex chromosome linkage of chicken and duck type I interferon genes: further evidence of evolutionary conservation of the Z chromosome in birds.

Type I interferons (IFNs) are a family of proteins that are predominantly expressed in response to viral infection. Two serologically distinct forms of type I IFN, designated ChIFN1 and ChIFN2, have recently been recognized in the chicken. ChIFN1 is encoded by a cluster of ten or more intronless genes, whereas ChIFN2, whose primary sequence is 57% identical, is encoded by a single intronless gene. By fluorescence in situ hybridization we now demonstrate that the genes for ChIFN1 and ChIFN2 are all located on the short arm of the chicken Z chromosome. This assignment was confirmed by results that showed that DNA from male (ZZ) chickens yielded approximately twofold stronger Southern blot signals with ChIFN1 and ChIFN2 hybridization probes than DNA from females (ZW). Attempts to determine differences in IFN production between male and female chickens failed owing to a high degree of variation in virus-induced IFN expression between individuals of both sexes. Sex linkage of IFN genes was also observed in domestic ducks: fluorescence in situ hybridization of duck metaphase chromosomes with a duck type I IFN probe was confined to the terminal region of the long arm of the Z chromosome. Thus, in contrast to mammals, which have their IFN genes on autosomes, birds have the type I IFN genes on the sex chromosome.

Activation of bcl-2 suppressible 40 and 44 kDa p38-like kinases during apoptosis of early and late B lymphocytic cell lines.

Activation of several different kinases characterizes the induction of apoptosis. Abelson virus transformed pre-B lymphocytes undergo apoptosis within 24 h of serum deprivation, PKA activation or gamma-irradiation, and the activity of two kinases of ca. 40 and 44 kDa is specifically induced during this apoptotic process. Bcl-2 expression prevents both apoptosis and the induction of these kinases. Immunologic and substrate similarities indicate that these kinases are related to the p38 family of MAP kinases. More mature cells of the B lymphocytic lineage, plasmacytomas, also exhibit induction of these kinases when apoptosis is induced by withdrawal of serum or IL-6. Treatment of the pre-B cells with ICE protease inhibitors when apoptotic stimuli are delivered prevents induction of the kinase activity, and partially inhibits apoptosis. These findings indicate that the induction of these 40 and 44 kDa p38 related kinases is a common feature of apoptosis in mouse B lymphocytic cells and may represent a step downstream of ICE proteases in the signal cascade that leads to programmed cell death.