Mary Sawyer

Experimental bluetongue and epizootic hemorrhagic disease virus infection in California black-tailed deer.

Four adult black-tailed deer (Odocoileus hemioneus columbianus) and five fawns were inoculated with bluetongue virus (BTV) and one adult deer was inoculated with epizootic hemorrhagic disease (EHD) virus to produce clinical signs and lesions of hemorrhagic disease. Serologic response was monitored using the agar gel immunodiffusion (AGID) test and the competitive enzyme-linked immunosorbent assay (C-ELISA). Embryonating chicken eggs and vero cells were used to detect viremia. No animal exhibited clinical or pathologic signs of hemorrhagic disease. Bluetongue viremia was detected as early as 2 days post-inoculation (DPI-2) and in some animals, persisted until at least DPI-12. The earliest detection of BTV antibodies using the AGID was DPI-8. Two adult deer remained seropositive for BTV antibodies for >9 mo and 1 yr, respectively, using both the AGID and C-ELISA tests. We observed cross reactions between BT and EHD antibodies using the AGID tests. Also, the AGID test did not consistently detect exposure to BTV. Viremia was not detected in the deer inoculated with EHD although this animal was AGID positive between DPI-6 and DPI-49.

Peroxidase-labeled primary antibody method for detection of pestivirus contamination in cell cultures

Contaminating bovine viral diarrhea (BVD) virus in cell cultures and in fetal bovine serum (FBS) is a well recognized problem. This study describes a direct peroxidase (DP) labeled primary antibody method for detection of pestivirus antigens in cell culture that is simple and reliable. Using this method, most bovine and porcine cell cultures, a cat lung, four mosquito and two monkey cell cultures were found contaminated with BVD virus. The rodent and human cell cultures tested were negative by this method for BVD virus.

Antimicrobial properties of the chelating agent EDTA on streptococcal bovine mastitis isolates

To determine the efficacy of the chelating agent EDTA on microbial growth, separate cultures of two streptococcal bovine mastitis isolates, Streptococcus agalactiae and Streptococcus uberis, were exposed to known concentrations of EDTA. Bacterial cultures of 10(8) CFU/ml were exposed to concentrations of EDTA ranging from 30 to 100 mM in an in-vitro-milk environment. Multiple replications of cultures exposed to EDTA were plated during a two-hour time course. A concentration of 100 mM EDTA resulted in a 90% reduction of S. agalactiae and a 99% reduction of S. uberis. Under these experimental conditions, EDTA treatments in cultures of both isolates exhibited from 1 to 2 log reductions suggesting that EDTA is a potentially effective antimicrobial against streptococcal isolates implicated in causing bovine mastitis.

Overcoming RNA Inhibition in the Fluorescent Polymerase Chain Reaction Assay to Enhance Detection of Bovine DNA in Cattle Feeds

The practice of incorporating mammalian protein in ruminant feeds was banned in the United States in 1997 as a measure to avoid transmission of bovine spongiform encephalopathy (BSE). A sensitive means of identifying the banned additives in feeds would be by detection of species-specific DNA using the polymerase chain reaction (PCR). However, problems may arise in the PCR due to the presence of inhibitory substances. Using human DNA as an internal PCR control, inhibitory substances were evident in the DNA extraction products of cattle feeds. The results of heating experiments excluded enzymes as a cause of inhibition, and spectrophotometric calculations suggested the possibility of RNA contamination. Co-electrophoresis of untreated and RNAse digested extracts confirmed the presence of RNA in the undigested product. Seven cattle feeds were spiked with predetermined amounts of bovine meat and bone meal (BMBM). The DNA extracted products were treated with RNAse and the bovine specific mitochondrial DNA (B-mtDNA) was amplified by PCR. The minimum level of detection of B-mtDNA was influenced by RNAse treatment and feed composition. RNAse treatment decreased false-negative results overall by 75%. False-negative results were decreased 100% in the higher BMBM concentrations and 50% in the lower BMBM concentrations. Also, each cattle feed was spiked to attain a 2% wt/wt concentration with each swine, fish, sheep, or poultry product, or cattle dried blood. Amplification of B-mtDNA occurred only with the cattle dried blood and only in three feeds in which B-mtDNA was detected at the only level tested (2%). A commercial immunochromotographic assay (Neogen) detected the spiked BMBM in only one of the seven feeds and only at the upper concentration (1%).

Detection of Rotavirus in Fecal Samples from Calves by a Cell Culture Indirect Immunofluorescence, an Ag-Capture ELISA, a Tissue Culture ELISA, and a Commercial Ag-Capture ELISA:

8 Rotavirus-caused diarrheas cannot be diagnosed solely on clinical signs; therefore, a rapid, simple, and sensitive laboratory detection method for rotavirus would be of a great diagnostic value. Several assays, including electron micros- copy (EM), have been developed for the rapid detection of rotaviruses or their antigens in stool samples of humans and animals. Because antigen-capture ELISAs (Ag-capture ELI- SAs) detect fragments of viral proteins in addition to intact virions, the purpose of this investigation was to evaluate an Ag-capture ELISA developed in our laboratory (Ag-C ELISA), a commercial ELISA kit a

Processing of wild caughtCulicoides and isolation of bluetongue virus using a mosquito cell line

Isolation of bluetongue virus from theCulicoides vector can provide valuable epidemiologic information. The methods described offer a practical procedure that ensures the preservation of the virus during field processing of the collection and an assay system that utilizes C6/36 cells, a clone derived fromAedes albopictus. The C6/36 cell line affords a sensitive isolation system and a practical virus detection system suitable for mass screening of insect pools.