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...

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...

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.Rotavirus, a member of the Reoviridae family, is an important cause of gastroenteritis in young children, calves, monkeys, chickens, pigs, sheep, and horses (1,14). It is nonenveloped and has double-shelled capsids surrounding a genome of 11 double-stranded RNA segments. Seven serological groups of rotavirus, A to G, have been identified, but only groups A, B, C, D, and G have been characterized well (15). Each group can be differentiated by polyacrylamide gel electrophoretic mobilities (2, 23).Among the seven serogroups, group A rotavirus has been studied in greatest detail, and it is the serogroup most commonly found in cattle worldwide. The virus is composed of a core surrounded by VP6, the major inner capsid protein. The outer capsid layers of infectious bovine rotavirus (BRV) particles contain two proteins, VP4 and VP7. The VP4 (P) types are spike protein encoded by RNA segment 4 (19, 21). They constitute important outer capsid proteins with various functions such as hemagglutinating activity (22) and neutralization activity (10,25,37), and when cleaved by trypsin into VP5 and VP8, they enhance the infectivity of the virus. There is evidence that rotavirus VP4 sequences are diverse (32). Using monoclonal antibodies (MAbs) against VP4, diversity has been shown in the amino acid sequences of epitopes that are critical for cross-reaction and neutralization of rotaviruses (18,19,22,33). Both VP4 and VP7 are associated with stimulation of serotype-specific antibodies and in vivo protection. Serotypes 1 to 4 of VP7 are glycosylated (6). Proteins other than VP4 and VP7, such as VP6, associated with stimulation of serotypespecific antibodies, may participate in protection against BRV infection; however, neutralizing antibodies in vitro have been shown to be specific against VP4 and VP7. Protection against rotavirus infection appears to rely mainly on stimulation of neutralizing antibodies against the outer capsid proteins, VP4 and VP7 (27).Many established protocols and commercial kits are available to detect rotavirus infection for human diagnostic medical applications including electron microscopy and enzyme-linked immunosorbent assay (E...