Cindy Chard-Bergstrom

Development, Evaluation, and Application of Lateral-Flow Immunoassay (Immunochromatography) for Detection of Rotavirus in Bovine Fecal Samples

ABSTRACT 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.

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

ABSTRACT 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.

Factors Affecting Isolation and Propagation of Bovine Coronavirus in Human Rectal Tumor-18 Cell Line

Bovine coronavirus (BCV) is an important cause of calf enterocolitis and respiratory disease. It is the second major cause of viral diarrhea in calves, with rotavirus being the first. At the Wisconsin Animal Health Laboratory during 1993-1994, BCV was detected in 93 cases of calf scours out of 1,058 bovine fecal samples examined by direct electron microscopy. Electron microscopy is used commonly for the diagnosis of enteric viruses, including BCV. The advantages of electron microscopy are that diagnosis can be made rapidly and multiple pathogens, a common feature in enteritis, can be detected simultaneously. Using electron microscopy, more than a dozen novel enteric viruses have been described in the last 2 decades. However, electron microscopy has some limitations; approximately 1 million viral particles should be present to detect a virus by electron microscopy. Thus, it lacks sensitivity and can lead to false-negative results. In addition, some viruses, especially coronaviruses, can be confused morphologically with nonviral particles such as intestinal brush border epithelium and with other morphologically similar viruses, leading to false-positive results. Virus isolation is not commonly used for the diagnosis of BCV. However, 1 advantage is that the virus propagated in cell culture can be used for further antigenic and genomic characterization. To improve BCV isolation from clinical samples, factors affecting its isolation and propagation in human rectal tumor18 (HRT18) cell line were investigated. In a previous study, 10 HRT-18 was found to be a suitable cell line for BCV isolation. In this study, BCV propagated in vitro showed a change in hemagglutination pattern from that of the BCV from the original clinical samples. It is not known if this change correlates with changes in antigenicity and immunogenicity of the virus. HRT-18 and human colon tumor-8 (HCT-8) cells are derived from adenocarcinomas of human rectum and colon, respectively. 16 The 51 samples included in this study were provided by the Wisconsin Animal Health Laboratory (WAHL), Madison (n = 27), the California Veterinary Diagnostic Laboratory (CVDL), San Bernardino (n = 6), and another diagnostic laboratory (referred to as VDL for confidentiality) (n = 18). Samples were obtained as 20% fecal suspensions in phosphate-buffered saline (PBS) (pH 7.2), as

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

ABSTRACT 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.

Immunohistochemical Demonstration of Francisella Tularensis in Lesions of Cats with Tularemia

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.

Development, Characterization, and Diagnostic Applications of Monoclonal Antibodies against Bovine Rotavirus

ABSTRACT 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.

Plaque Variations in Clinical Isolates of Bovine Coronavirus

Bovine coronavirus (BCV) causes enterocolitis and respiratory disease in young calves and may cause chronic diarrhea (winter dysentery) in adult cattle. To understand the molecular basis of tissue tropism and virulence, and to study biological differences between clinical isolates, it is important to plaque purify BCV. Plaque assay can also be used to study the effect(s) of antiviral drugs, such as hygromycin B, on BCV replication in vitro. Moreover, there is an urgent need to select better vaccine candidates and develop an effective modified live virus vaccine against BCV, and candidate vaccine isolates must be plaque purified for characterization. Because isolates of BCV are obtained from fecal samples that contain a wide variety of bacteria and viruses, plaque purification is essential. This paper describes the development of a plaque assay to study plaque variation in recent isolates of BCV (1993-1994) and to study the antiviral effects of hygromycin B on BCV in vitro. Human rectal tumor-18 (HRT-18) cells were plated in 6-mm tissue culture dishes and grown in minimum essential medium (MEM) with 10% fetal calf serum, L-glutamine, and antibiotics (Kapil S, Donnelly C: 1994, Abstr Proc 75th Conf Res Workers Anim Dis, P77). After 3-4 days, confluent monolayers were washed with calciumand magnesium-free phosphate-buffered saline (CMF-PBS). BCV isolates were obtained from calves with a history of diarrhea. Approximately 250 μl of virus dilutions (10 -10) was added to these plates. For BCV adsorption, the plates were hand-rocked every 5-10 minutes for 1 hour. Monolayers were washed with CMF-PBS and overlaid with 1% agarose in MEM containing trypsin (5 μg/ml) and pancreatin (5 μg/ml). To study the effect of hygromycin B on BCV replication the drug was incorporated in the agarose at concentrations of 0.1 mM, 0.25 mM, 0.5 mM, and 1 mM. Plates were incubated in an inverted position in a humid chamber at 37 C for 3-5 days. To count and measure the plaques, the plates were held against a bright source of light. Since our protocol gave clearly visible plaques, it was not necessary to stain the plates. Viral titers were calculated on the basis of dilutions that gave about 2530 isolated plaques/well. To confirm the identities of the plaques, the monolayers were fixed with 5 ml of 10% neutral buffered formalin per plate. In preliminary experiments, we compared 100% methanol, 100% ethanol, 5% glutaralde-

Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung.

To gain insight into the mechanism by which angiotensin II type 2 receptor (AT2) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT2 single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT2 receptor protein. The specificity of the antibodies was verified using AT2 over-expressing COS-7 cells and AT2 naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT2 and AT1 immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors’ expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.