Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry.

Loop-mediated isothermal amplification (LAMP) is a simple, powerful state-of-the-art gene amplification technique used for the rapid diagnosis and early detection of microbial diseases. Many LAMP assays have been developed and validated for important epizootic diseases of livestock. We review the LAMP assays that have been developed for the detection of 18 viruses deemed notifiable of ruminants, swine and poultry by the World Organization for Animal Health (OIE). LAMP provides a fast (the assay often takes less than an hour), low cost, highly sensitive, highly specific and less laborious alternative to detect infectious disease agents. The LAMP procedure can be completed under isothermal conditions so thermocyclers are not needed. The ease of use of the LAMP assay allows adaptability to field conditions and works well in developing countries with resource-limited laboratories. However, this technology is still underutilized in the field of veterinary diagnostics despite its huge capabilities.

Mechanism of cell death during infectious salmon anemia virus infection is cell type-specific.

Infectious salmon anemia virus (ISAV) is a very important fish virus in the Northern hemisphere and there is continued interest in understanding the mechanisms of its pathogenesis and persistence in fish. In this study, the permissive fish cell lines SHK-1, CHSE-214 and TO were used to determine whether ISAV-induced cytopathic effect (CPE) is due to apoptosis or necrosis. Characteristic apoptotic DNA fragmentation was observed only in ISAV-infected SHK-1 and CHSE-214 cells. Apoptosis in ISAV-infected SHK-1 cells was confirmed by fragment end-labelling assay, suggesting that CPE in these cells is associated with apoptosis. ISAV-infected TO cells did not undergo apoptosis, but showed leakage of high-mobility group 1 (HMGB1) protein from the nucleus, which is characteristic of cells undergoing necrosis; this suggests that CPE in these cells is associated with necrosis. ISAV-infected SHK-1 cells did not show leakage of HMGB1 protein. Infection with two different strains of ISAV showed that induction of apoptosis was correlated with the appearance of CPE in SHK-1 cells. ISAV-induced apoptosis was inhibited by a pan-caspase inhibitor, Z-VAD-fmk, indicating a caspase-activation pathway. The ISAV putative PB2 protein and proteins encoded by RNA segment 7 bound caspase-8 specifically in vitro, suggesting that these viral proteins may have a role in ISAV-induced apoptosis. These findings demonstrate for the first time that the mechanism of cell death during ISAV infection is dependent on the cell type, which may have implications for ISAV pathogenesis and persistence.

Use of epitope mapping to identify a PCR template for protein amplification and detection by enzyme-linked immunosorbent assay of bovine herpesvirus type 1 glycoprotein D.

Infection with bovine herpesvirus type 1 (BHV-1) occurs worldwide and causes serious economic losses due to the deaths of animals, abortions, decreased milk production, and loss of body weight. BHV-1 is frequently found in bovine semen and is transmitted through natural service and artificial insemination. The detection of BHV-1 in bovine semen is a long-standing problem in veterinary virology which is important in disease control schemes. In the present study, ordered deletions of the full-length BHV-1 glycoprotein open reading frame were used to identify an epitope recognized by a specific monoclonal antibody (MAb). A glycoprotein D fragment containing this epitope was then amplified using an in vitro protein amplification assay developed previously (J. Zhou, J. Lyaku, R. A. Fredrickson, and F. S. Kibenge, J. Virol. Methods 79:181-189, 1999), and the resulting peptide was detected by indirect enzyme-linked immunosorbent assay (ELISA) with the specific MAb. This method detected 0.0395 50% tissue culture infective dose of BHV-1 in raw bovine semen, which was 1,000-fold more sensitive than traditional PCR. We therefore conclude that this in vitro protein amplification assay combined with ELISA has superior sensitivity for direct virus detection in clinical samples.