Andrea Gröne

Chronic enteropathies in dogs: evaluation of risk factors for negative outcome.

HYPOTHESIS Certain variables that are routinely measured during the diagnostic evaluation of dogs with chronic enteropathies will be predictive for outcome and a new clinical disease activity index incorporating these variables can be applied to predict outcome of disease. ANIMALS Seventy dogs were entered into a sequential treatment trial with elimination diet (FR, food-responsive group) followed by immunosuppressive treatment with steroids if no response was seen with the dietary trial alone (ST, steroid-treatment group). A 3rd group consisted of dogs with panhypoproteinemia and ascites (PLE, protein-losing enteropathy) that were treated with immunosuppressive doses of steroids. METHODS Three years of follow-up information was available for all dogs. Clinicopathologic variables were tested for their ability to predict negative outcome, defined as euthanasia due to refractoriness to treatment. Different scoring systems including different combinations of these variables were evaluated using receiver operating characteristic (ROC) curves. RESULTS Thirteen of 70 (18%) dogs were euthanized because of intractable disease. Univariate analysis identified a high clinical activity index, high endoscopic score in the duodenum, hypocobalaminemia (<200 ng/L) and hypoalbuminemia (<20 g/L) as risk factors for negative outcome. CONCLUSIONS AND CLINICAL IMPORTANCE Based on the factors identified by logistic regression and ROC curve analysis, a new clinical scoring index (CCECAI) was defined that predicts negative outcome in dogs suffering from chronic enteropathies.

Binding of Avian Coronavirus Spike Proteins to Host Factors Reflects Virus Tropism and Pathogenicity

ABSTRACT The binding of viruses to host cells is the first step in determining tropism and pathogenicity. While avian infectious bronchitis coronavirus (IBV) infection and avian influenza A virus (IAV) infection both depend on α2,3-linked sialic acids, the host tropism of IBV is restricted compared to that of IAV. Here we investigated whether the interaction between the viral attachment proteins and the host could explain these differences by using recombinant spike domains (S1) of IBV strains with different pathogenicities, as well as the hemagglutinin (HA) protein of IAV H5N1. Protein histochemistry showed that S1 of IBV strain M41 and HA of IAV subtype H5N1 displayed sialic acid-dependent binding to chicken respiratory tract tissue. However, while HA bound with high avidity to a broad range of α2,3-linked sialylated glycans, M41 S1 recognized only one particular α2,3-linked disialoside in a glycan array. When comparing the binding of recombinant IBV S1 proteins derived from IBV strains with known differences in tissue tropism and pathogenicity, we observed that while M41 S1 displayed binding to cilia and goblet cells of the chicken respiratory tract, S1 derived from the vaccine strain H120 or the nonvirulent Beaudette strain had reduced or no binding to chicken tissues, respectively, in agreement with the reduced abilities of these viruses to replicate in vivo. While the S1 protein derived from the nephropathogenic IBV strain B1648 also hardly displayed binding to respiratory tract cells, distinct binding to kidney cells was observed, but only after the removal of sialic acid from S1. In conclusion, our data demonstrate that the attachment patterns of the IBV S proteins correlate with the tropisms and pathogenicities of the corresponding viruses.

Immunohistochemical Localization of Clostridium perfringens β2-Toxin in the Gastrointestinal Tract of Horses

Clostridia-associated intestinal disease in horses was generally reported to be due to infection with Clostridium perfringens type A, which harbors the cpa-encoded α-toxin. A recent study demonstrated a high incidence of β2-toxigenic C. perfringens in horses suffering or dying from typhlocolitis, suggesting that this novel type of C. perfringens might play an important role in typhlocolitis and possibly other equine intestinal diseases. A retrospective study was conducted to assess the presence of the β2-toxin in tissues of the equine gastrointestinal tract. Monospecific polyclonal antibodies against recombinant β2-toxin were produced in rabbits and used to demonstrate the β2-toxin in sections of the gastrointestinal tract by immunohistochemical methods. Sections from 69 horses were stained and β2-toxin was observed immunohistochemically in 40 animals. Sections from the stomach, small intestine, and large intestine were positive. Immunopositivity for β2-toxin was significantly associated with presence of β2-toxigenic bacteria. This investigation demonstrates local production of β2-toxin and suggests that immunohistochemistry using antitoxin antibodies represents a useful diagnostic method in those cases where isolation of bacteria and polymerase chain reaction typing is not feasible. Although the association between the presence of β2-toxin and development of gastrointestinal disease in horses remains uncertain, the findings of this study indicate that the potential causal relationship warrants further investigation.

Perinuclear Antineutrophilic Cytoplasmic Antibody and Response to Treatment in Diarrheic Dogs with Food Responsive Disease or Inflammatory Bowel Disease

The goal of this study was to investigate the correlation between perinuclear antineutrophilic cytoplasmic antibody (pANCA) and clinical scores before and after treatment in diarrheic dogs with food-responsive disease (FRD) or inflammatory bowel disease (IBD). pANCA serology was evaluated prospectively by indirect immunofluorescence in 65 dogs with signs of gastrointestinal disease, and if positive, pANCA antibody titers were determined. Thirty-nine dogs with FRD responded to a novel diet, and 26 dogs with IBD were treated with corticosteroids. The severity of clinical signs was scored by means of a canine IBD activity index (CIBDAI). At initial examination, a significantly (P = .002) higher percentage of dogs were pANCA-positive in the FRD group (62%) compared with the IBD group (23%). pANCA titers were significantly higher (P = .003) before treatment in the FRD group (median titer 100) compared with the IBD group (median titer 1). However, there was no difference in pANCA titers between the groups after respective treatments because dogs in the IBD group had a significant increase in pANCA titer after treatment. The CIBDAI score decreased significantly (P < .001) after treatment in both groups (74% moderate to severe in FRD dogs before versus 8% after treatment; 85% moderate to severe in IBD dogs before versus 32% after treatment). There was no correlation between pANCA status in FRD or IBD dogs before treatment and scores for CIBDAI, endoscopy, or histopathology before or after treatment, except for the endoscopic duodenal score in dogs with FRD after treatment (P = .03). A positive pANCA test before therapy may aid in the diagnosis of FRD.

Mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus

Abstract The infection of the avian coronavirus infectious bronchitis virus (IBV) is initiated by the binding of the spike glycoprotein S to sialic acids on the chicken host cell. In this study we identified the receptor-binding domain (RBD) of the spike of the prototype IBV strain M41. By analyzing the ability of recombinantly expressed chimeric and truncated spike proteins to bind to chicken tissues, we demonstrate that the N-terminal 253 amino acids of the spike are both required and sufficient for binding to chicken respiratory tract in an α-2,3-sialic acid-dependent manner. Critical amino acids for attachment of M41 spike are present within the N-terminal residues 19–69, which overlap with a hypervariable region in the S1 gene. Our results may help to understand the differences between IBV S1 genotypes and the ultimate pathogenesis of IBV in chickens.

The occurrence of cpb2-toxigenic Clostridium perfringens and the possible role of the β2-toxin in enteric disease of domestic animals, wild animals and humans.

The virulence of Clostridium perfringens, a bacterium causing enteritis and enterotoxaemia in domestic and wild animals and humans, results largely from its ability to produce toxins. In 1997, an unknown toxin of C. perfringens, the beta2-toxin, and its encoding gene cpb2 were described. Since that time numerous studies have been published dealing with a possible association of cpb2-harbouring strains of C. perfringens and the occurrence of enteric disease in domestic and wild animals and humans. This article offers an overview of the current literature on the spread and pathological significance of cpb2-harbouring C. perfringens. Unambiguous conclusions on the prevalence of cpb2 and the contribution of beta2-toxin to the disease cannot be drawn from existing studies but in some animal species a strong correlation between the presence of cpb2-harbouring C. perfringens, the beta2-toxin and enteric disease has been reported.

Clinical Signs, Histology, and CD3-Positive Cells before and after Treatment of Dogs with Chronic Enteropathies

BACKGROUND Histopathology is widely used for the diagnosis of inflammatory bowel disease in dogs. Variations in lesions and unavailability of uniform grading systems limit the usefulness of histologic examination. HYPOTHESIS CD3 cell numbers in chronic enteropathies of dogs correlate with clinical activity of the disease and with severity of histopathologic changes. ANIMALS Nineteen client-owned dogs examined because of chronic diarrhea, vomiting, or both. METHODS Samples of duodenal and colonic mucosa were collected endoscopically before and after treatment. Dogs that responded to a hypoallergenic diet were grouped as food-responsive diarrhea dogs (FRD, n=10). Dogs with no clinical improvement after 10 days of treatment then received prednisolone (immunosuppressive doses) and were grouped as steroid-responsive diarrhea dogs (SRD, n=9). Histopathologic assessment with a standardized grading system was performed retrospectively on the intestinal samples. Histologic score, total number of infiltrating cells, and CD3-positive cells were counted and compared with the clinical scoring. RESULTS No statistically significant difference was detected among histologic grading, total number of cells in the lamina propria, and T-cell numbers in biopsies before and after treatment in either group (FRD and SRD). CONCLUSIONS AND CLINICAL IMPORTANCE Currently used histopathologic grading scores, total numbers of cells, and numbers of CD3-positive cells did not allow differentiation between FRD and SRD and did not correlate with clinical response to therapy. Based on these results, new grading scores assessing other criteria than total cell numbers and CD3-positive cells should be evaluated in the future.

Antibiotic‐induced expression of a cryptic cpb2 gene in equine β2‐toxigenic Clostridium perfringens

The cpb2 gene of β2‐toxigenic Clostridium perfringens isolated from horses, cattle, sheep, human and pigs was sequenced. The cpb2 gene of equine and other non‐porcine isolates differed from porcine isolates by the absence of an adenine in a poly A tract immediately downstream of the start codon in all non‐porcine C. perfringens strains. This deletion involved formation of a cryptic gene harbouring a premature stop codon after only nine amino acid codons, while the full β2‐toxin protein consists of 265 amino acids. Immunoblots carried out with antibodies directed against a recombinant β2‐toxin showed the absence of expression of the β2‐toxin in equine and the other non‐porcine strains under standard culture conditions. However, treatment of C. perfringens with the aminoglycosides gentamicin or streptomycin was able to induce expression of the cpb2 gene in a representative equine strain of this group, presumably by frameshifting. The presence of the β2‐toxin was revealed by immunohistology in tissue samples of small and large intestine from horses with severe typhlocolitis that had been treated before with gentamicin. This result may explain the finding that antibiotic treatment of horses affected by β2‐toxigenic C. perfringens leads to a more accentuated and fatal progression of equine typhlocolitis. Clinical observations show a reduced appearance of strong typhlocolitis in horses with intestinal complications admitted to hospital care since the standard use of gentamicin has been abandoned. This is the first report on expression of a bacterial toxin gene by antibiotic‐induced ribosomal frameshifting.

Ranavirus-associated mass mortality in wild amphibians, the Netherlands, 2010: a first report.

In 2010, a mass die-off of over 1000 wild water frogs (Pelophylax spp.) and at least 10 common newts (Lissotriton vulgaris) occurred in a pond in The Netherlands. Haemorrhagic disease with hepatomegaly and splenomegaly was evident. Microscopically, multiple organs presented cells with multifocal intracytoplasmic inclusion bodies, in which ranavirus-like particles were demonstrated ultrastructurally. All specimens examined tested positive for ranavirus by PCR. The sequence obtained showed a 100% identity with the one deposited for common midwife toad virus (CMTV). This is the first report of ranavirus-associated mortality in wild amphibian populations in The Netherlands. It is also the first time CMTV or a CMTV-like virus has been reported in these two species in the adult stage and outside of Spain.

Proteasome Immunosubunits Protect against the Development of CD8 T Cell-Mediated Autoimmune Diseases

Exposure of cells to inflammatory cytokines induces the expression of three proteasome immunosubunits, two of which are encoded in the MHC class II region. The induced subunits replace their constitutive homologs in newly formed “so-called” immunoproteasomes. Immunosubunit incorporation enhances the proteasome’s proteolytic activity and modifies the proteasome’s cleavage-site preferences, which improves the generation of many MHC class I-presented peptides and shapes the fine specificity of pathogen-specific CD8 T cell responses. In this article, we report on a second effect of immunoproteasome formation on CD8 T cell responses. We show that mice deficient for the immunosubunits β5i/low molecular mass polypeptide (LMP7) and β2i/multicatalytic endopeptidase complex-like–1 develop early-stage multiorgan autoimmunity following irradiation and bone marrow transplantation. Disease symptoms are caused by CD8 T cells and are transferable into immunosubunit-deficient, RAG1-deficient mice. Moreover, using the human Type 1 Diabetes Genetics Consortium MHC dataset, we identified two single nucleotide polymorphisms within the β5i/LMP7-encoding gene sequences, which were in strong linkage disequilibrium, as independent genetic risk factors for type 1 diabetes development in humans. Strikingly, these single nucleotide polymorphisms significantly enhanced the risk conferred by HLA haplotypes that were previously shown to predispose for type 1 diabetes. These data suggested that inflammation-induced immunosubunit expression in peripheral tissues constitutes a mechanism that prevents the development of CD8 T cell-mediated autoimmune diseases.