Ovine adenovirus serotype 7-associated mortality in lambs in the United States.

Adenoviral infections were diagnosed in three neonatal lambs that died spontaneously, and no other etiologic agents were identified. Clinical signs were anorexia, weakness, abdominal distention, and sudden death. Microscopic lesions consisted of multifocal necrotizing hepatitis, multifocal subacute interstitial nephritis, and loss of enterocytes from intestinal villi. Adenovirus inclusions were identified by light microscopy in the kidneys only. Adenoviral antigen, however, was identified in the liver, kidney, and intestine of the lambs by immunohistochemical techniques. An ovine adenovirus serotype 7, not previously isolated from sheep in the United States, was characterized from these lambs.

Evaluation of a latex agglutination kit (Virogen Rotatest) for detection of bovine rotavirus in fecal samples.

The performance of the Virogen Rotatest latex agglutination test (LAT) was evaluated for detection of bovine rotavirus antigen. Sixty-three fecal samples from diarrheic calves were collected from November 1999 to May 2000 and screened by LAT, the Rotazyme II enzyme-linked immunosorbent assay (ELISA), and virus isolation (VI) followed by an anti-rotavirus fluorescent-antibody (FA) test to detect the presence of group A rotavirus antigen. Of the 63 samples screened by VI-FA, 33 (58%) tested positive for rotavirus antigen. When the results from the LAT were compared to those from VI-FA, the "gold standard" for detection of bovine rotavirus in fecal samples, the sensitivity and specificity were found to be 87.8 and 73.3%, respectively. Latex agglutination compared with ELISA (the reference method) showed 100% sensitivity and 96.3% specificity, and when ELISA was compared with VI, the sensitivity was 84.8% and the specificity was 73.3%. Latex agglutination is easy to perform in a short time and does not require expensive equipment or skilled personnel, and the reagents have long shelf lives. These factors make the LAT suitable and highly efficient for use in a clinical laboratory as a rapid screening test for bovine rotavirus.

Development of a recombinant nucleoprotein-based enzyme-linked immunosorbent assay for quantification of antibodies against porcine reproductive and respiratory syndrome virus.

A rapid, inexpensive enzyme-linked immunosorbent assay (ELISA) to quantitate antibodies to porcine respiratory and reproductive syndrome virus (PRRSV) in serum was developed using a recombinant PRRSV nucleoprotein (rN). The sensitivity (85.3%) and specificity (81.7%) of the Kansas State University ELISA were good, correlating well (82.4%) with the IDEXX HerdChek ELISA.

Development, characterization, and diagnostic applications of monoclonal antibodies against bovine rotavirus.

Hybridomas secreting monoclonal antibodies (MAbs) against the Nebraska calf diarrhea strain of bovine rotavirus (BRV) were characterized. Indirect fluorescent-antibody assay, immunodot assay, and immunoprecipitation were used to select hybridomas that produced anti-BRV MAbs. Seven of the MAbs were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot assay to be reactive with the BRV outer capsid protein, VP7, which has a molecular mass of 37.5 kDa. None of the seven MAbs were reactive with canine rotavirus, bovine coronavirus, or uninfected Madin-Darby bovine kidney cells. Two clones, 8B4 (immunoglobulin G2a [IgG2a]) and 2B11 (IgG1), were found suitable for use in an antigen capture enzyme-linked immunosorbent assay for detecting BRV in bovine fecal samples. Both were subtype A specific (G6 subtype) but did not react with all isolates of BRV group A.

Production, characterization, and uses of monoclonal antibodies against recombinant nucleoprotein of elk coronavirus.

This is the first report of the production of monoclonal antibodies against elk coronavirus. The nucleoprotein gene of elk coronavirus was amplified by PCR and was cloned and expressed in a prokaryotic expression vector. Recombinant nucleocapsid protein was used to immunize mice for the production of hybridomas. Twelve hybridomas that produced monoclonal antibodies against the nucleocapsid protein of elk coronavirus were selected by an indirect fluorescent-antibody test, an enzyme-linked immunosorbent assay, and a Western blot assay. Ten of the monoclonal antibodies were of the immunoglobulin G1 (IgG1) isotype, one was IgG2a, and one was IgM. All had kappa light chains. By immunohistochemistry four monoclonal antibodies detected bovine coronavirus and elk coronavirus in formalin-fixed intestinal tissues. Antinucleoprotein monoclonal antibodies were found to be better at ruminant coronavirus detection than the anti-spike protein monoclonal antibodies. Because nucleoprotein is a more abundant antigen than spike protein in infected cells, this was not an unexpected finding.

Application of immunohistochemistry and in situ hybridization for detection of bovine coronavirus in paraffin-embedded, formalin-fixed intestines.

A monoclonal antibody (MAb) (Z3A5) against spike protein subunit of bovine coronavirus (BCV) reacted with the virus in formalin-fixed intestines in an immunoperoxidase test. We found an 88% correlation between immunohistochemistry with Z3A5 and in situ hybridization with a BCV nucleoprotein cDNA probe. MAb Z3A5 reacted with 90 BCV isolates from the United States and was an effective reagent for the diagnosis of BCV.

Immunohistochemistry of pancreatic islet cell tumors in the ferret (Mustela putorius furo).

Twenty-two pancreatic islet cell tumors and normal pancreatic islets from ferrets were evaluated by immunohistochemistry for expression of the peptide hormones insulin, somatostatin, glucagon, and pancreatic polypeptide (PP) and the neuroendocrine markers chromogranin A (CgA) and neuron-specific enolase (NSE). In normal pancreatic islets, the majority of cells stained strongly with CgA and NSE. A cells, B cells, D cells, and PP cells stained strongly with glucagon, insulin, somatostatin, and PP, respectively. All 22 tumors stained with CgA and NSE. The proportion of cells within tumors staining for CgA was variable, but more than half of the cells stained positively in 18 of the tumors. The intensity of staining for CgA was strongly (reactivity equivalent to or greater than normal islet cells in adjacent tissue) in 11 moderate in six, and weak in five of the tumors. All tumors stained for NSE, with > or = 50% of the cells staining in 21 of the tumors, and the intensity of staining was strong in 18 of the tumors. Twenty of 22 tumors stained positively for insulin. with > or = 50% of the cells staining in 19 of them. The intensity of staining for insulin was strong in 12, moderate in seven, and weak in one of the tumors. Approximately < or = 1% of the cells in 15 of 22 tumors stained for somatostatin, five tumors stained for pancreatic polypeptide, and three tumors stained for glucagon. These data indicate that the majority of islet cell tumors of ferrets express immunohistochemically detectable insulin. CgA and NSE are both useful general markers for such tumors, including those that are insulin negatives. Commercially available antisera to CgA, NSE, insulin, glucagon, somatostatin, and PP work well in formalin-fixed, paraffin-embedded tissue for immunophenotyping islet cell tumors in the ferret.

Chromogranin A plasma concentration and expression in pancreatic islet cell tumors of dogs and cats.

OBJECTIVES: To describe expression of the neuroendocrine marker chromogranin A (CgA) in canine and feline pancreatic islet cell tumors and their metastases, and to evaluate plasma CgA concentration in dogs and cats with insulinoma. SAMPLE POPULATION: Paraffin-embedded tissues from 25 canine and 2 feline pancreatic islet cell tumors, 5 canine and 6 feline exocrine pancreatic tumors, and normal pancreatic tissue from 2 dogs and 2 cats. Heparinized plasma samples from 3 dogs and 2 cats diagnosed with insulinoma, and 10 control plasma samples from each species. PROCEDURE: Immunohistochemical analysis was performed on the 42 tissue specimens, using antisera against CgA, neuron-specific enolase, insulin, somatostatin, glucagon, and pancreatic polypeptide. The 25 plasma samples were evaluated, using a soluble-phase, double-antibody, equilibrium radioimmunoassay directed against the amino- and carboxy-terminal peptides of bovine CgA. RESULTS: Chromogranin A expression was found in 76% of canine and 2 of 2 feline pancreatic islet cell tumors. Of 7 animals with CgA immunoreactivity in primary tumors, 6 also had CgA immunostaining of metastatic lesions. Plasma CgA concentration in 2 dogs with insulinoma (0.9, 1.0 ng/ml) exceeded the reference range established for 10 clinically normal control dogs (0.50 +/- 0.16 ng/ml). Feline plasma CgA samples had extensive nonspecific background immunoreactivity. CONCLUSIONS: Chromogranin A is a useful immunohistochemical marker for pancreatic tumors of neuroendocrine origin and their metastases. Plasma CgA concentration determined by radioimmunoassay was high in 2 dogs with insulinoma. CLINICAL RELEVANCE: Immunohistochemical staining of tissues or cytologic specimens for CgA and/or neuron-specific enolase may help distinguish masses of unknown origin as neuroendocrine in nature. Increase in plasma CgA concentration may be useful diagnostically for animals with suspected neuroendocrine tumors.