A lateral-flow immunoassay (LFT) was developed to detect bovine rotavirus in fecal samples. Using samples (n ‫؍‬ 74) from diarrheic calves, a comparison of the LFT with a commercial latex agglutination test (LAT) and transmission electron microscopy (EM) was conducted. When EM was used as the reference method, initial studies of 29 samples indicated 70 and 80% sensitivities of the LFT and LAT, respectively, with both being 100% specific. When the LAT was the reference test, the LFT was 75% sensitive and 91% specific. Additional specimens (n ‫؍‬ 45) were tested by the LFT and LAT alone, and results were identical for both methods.Rotaviruses are the major cause of severe gastroenteritis in human infants and animals worldwide (2, 5-9). Bovine rotavirus (BRV) is responsible for approximately 30% of calf enteritis (3); thus, accurate and rapid diagnostic tests are important for proper treatment and prevention. The objective of this study was to develop and evaluate a lateral-flow immunoassay (LFT) for rapid detection of BRV in fecal samples. Evaluation of the LFT was conducted by comparing its performance to that of transmission electron microscopy (EM) and the latex agglutination test (LAT).Seventy-four fecal samples from calves with acute diarrhea were tested. Fecal samples and supernatants of BRV-infected cell cultures previously tested by enzyme-linked immunosorbent assay (ELISA) with the Rotazyme II kit (Abbott Laboratories, Abbott Park, Ill.) were used as positive and negative controls for this study (1).For examination by EM, 10% (wt/vol) suspensions of calf feces were homogenized in 0.01 M phosphate-buffered saline (PBS) (pH 7) and were centrifuged at 1,500 ϫ g for 5 min. The supernatant was removed and centrifuged at 28,000 ϫ g for 1 h. A 35-l aliquot of pelleted material was treated with 0.7 ml of double-distilled water, 140 l of phosphotungstic acid (pH 7), and 35 l of 1% bovine serum albumin for 5 min and then was sprayed onto carbon-coated grids for viewing at ϫ30,000 magnification. The manufacturer's instructions for testing of human fecal samples were followed to test the bovine feces using the Virogen Rotatest (Wampole Laboratories, Cranbury, N.J.), a rapid LAT which utilizes latex particles coated with antibodies specific for group A rotavirus antigens.All components of the LFT system, including BRV antiserum, anti-mouse immunoglobulin G (IgG), antirotavirus antibody-gold conjugated, membrane-blocking reagent, and known positive and negative controls, were evaluated in a series of titration experiments to determine optimum concentrations for the assay. Criteria used in these experiments were the attainment of maximum signal strength for specific line formation (true-positive/true-negative result) with an absence of nonspecific line formation (false-positive/false-negative result) and low background staining. The colloidal gold particles (2 nm; BBI International, Cardiff, United Kingdom) and monoclonal antibody used to prepare the LFT indicator conjugate were handled following the manufacturer's inst...

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.

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.Rotaviruses are nonenveloped viruses belonging to the genus Rotavirus in the family Reoviridae. They are the major causes of dehydration and diarrhea in young children and many animal species. Group A rotaviruses are the major causes of enteric disease in calves (11,14,18,22,23,24). The classification of rotaviruses into serotypes is based on the identification of two outer proteins, VP4 and VP7 (16,19). Both VP4 and VP7 elicit the production of neutralizing antibodies shown to be protective against bovine rotavirus (BRV) infection in vivo and in vitro (4, 7, 9). VP7 serotypes (G types) can now be identified by enzyme immunoassay incorporating VP7-specific neutralizing monoclonal antibodies (16,18).Several tests are used routinely in diagnostic laboratories for the detection of rotavirus in fecal samples. These include enzyme-linked immunosorbent assay (ELISA) (2, 5, 13), electron microscopy, virus isolation (VI), passive hemagglutination, immunoelectrophoresis, and latex agglutination assays (6,8,9,10,20).In this study, we compared the Virogen Rotatest kit (Wampole Laboratories, Cranbury, N.J.), a latex agglutination test (LAT), with the Rotazyme II ELISA kit (Abbott Laboratories, Abbott Park, Ill.) and with VI for sensitivity and specificity of detection of BRV in fecal samples. VI followed by a fluorescent-antibody (FA) test was used as the "gold standard" method. MATERIALS AND METHODSFecal collection and preparation. Sixty-three fecal specimens obtained from calves with acute gastroenteritis were submitted to the Veterinary Diagnostic Laboratory at Kansas State University, Manhattan, between November 1999 and May 2000. These cases were from Kansas and Nebraska. The negative and positive control samples were from healthy animals and from cell culture supernatants that tested positive, respect...

An immunohistochemical test was developed and validated for detection of Francisella tularensis antigen in tissues of cats with fatal tularemia. Ten cases of naturally occurring tularemia in cats were positive both by isolation of F. tularensis and immunohistochemical identification of F. tularensis antigen. Nine additional cases with lesions typical of tularemia were positive for F. tularensis antigen, although bacterial cultures were not performed. Immunohistochemical identification of F. tularensis in formalin-fixed tissue is valuable for establishing a rapid etiologic diagnosis under circumstances where fresh tissues may not be available for isolation and identification of the organism.

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