Development of a DNA microarray-based multiplex assay of avian influenza virus subtypes H5, H7, H9, N1, and N2.

Outbreaks of highly pathogenic avian influenza have caused considerable economic losses in the poultry industry and have also resulted in human deaths since 2004. Rapid subtyping of highly pathogenic avian influenza viruses(HPAIVs) in clinical specimens is a prerequisite of prompt control of disease and prevention of its spreading. In this study, we describe development of a DNA microarray-based detection and subtyping of HPAIVs in field samples. DNA copies of matrix (M) protein genes for the H5, H7, and H9 subtypes of hemagglutinin (HA) and the N1 and N2 subtypes of neuraminidase (NA) were prepared by RT-PCR and specific primers and then spotted onto aldehyde slides to form DNA microarrays. The HPAIV samples to be tested were subjected to total RNA isolation, RT-PCR with universal primers and Cy3 labeling, and the obtained double-stranded DNAs (targets) were finally hybridized with DNA microarrays (probes). A fluorescent spot on the microarray, detected by scanning indicated positive hybridization, i.e. the involved subtype. The assay was specific as various heterologous viruses or HPAIVs of other subtypes tested were negative. No cross-hybridization among different subtypes could be detected. The assay was more sensitive than RT-PCR and chicken embryo inoculation and could be also used for field samples. Summing up, the assay has proved useful for simultaneous detection and differentiation of main epidemic HPAIV subtypes.

Development of an enzyme-linked immunosorbent assay for the diagnosis of Theileria sp. infection in sheep.

A rapid, sensitive and specific diagnostic method, an enzyme-linked immunosorbent assay (ELISA), was developed for the diagnosis of Theileria sp. infection in sheep; and optimal conditions were established, such as antigen concentration, serum dilution, coating time, Tween-20 concentration and conjugate. The results were analyzed by measuring the coefficient of variation (CV). Three sera titers (high, middle, low) were analyzed over the measurement range, resulting in a CV of around 10%, whereas a 30% variation is the maximum acceptable. The cut-off value was determined by the mean of a negative control plus three standard deviations. Cross-reaction was found only with Babesia ovis. However, this result may be questionable, because it cannot be excluded that these sheep were already infected with both Theileria sp. and B. ovis. The ELISA described in the present study proved to be a useful tool for studying the epidemiology of Theileria sp.