Many commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses to different avian influenza virus (AIV) subtypes. Developing an ELISA for specifically detecting the H5 antibody is the purpose of this study. Four monoclonal antibodies (Mabs) were raised using A/duck/Yunlin/04 (H5N2). They were confirmed as being specific to H5. Two of these antibodies showed hemagglutination inhibition (HI) activity using the HI test. Using immunodot blot assays, three Mabs recognized both Eurasian and American H5, whereas the other Mab recognized only the tested Eurasian H5 virus. When testing denatured H5 antigen, one of the Mabs lost its antigen binding activity using Western blotting. For detecting the H5 humoral response in serum, one monoclonal antibody was purified and labeled with horseradish peroxidase to set up a blocking ELISA. Chicken sera that blocked H5 Mab binding by > 29% were considered H5 antibody positive. Inhibition percentages for sera from chickens infected with other AIV subtypes, H1 to H15, were < 29%. This blocking ELISA was used for 478 field chicken serum samples. The results showed that the sensitivity and specificity of this ELISA were 98.3% (232/236) and 95.9% (232/242), respectively. This blocking ELISA could be used specifically for detecting the H5 humoral responses in chickens.
Rodent pinworms, Spironucleus muris and Tritrichomonas muris are the endoparasites that should be monitored and excluded from laboratory animal colonies. Nevertheless, traditional diagnostic methods may not efficiently detect and accurately demonstrate the endoparasite infestation status. In this study, we developed a multiplex PCR assay targeting the rRNA genes to simultaneously detect and differentiate five endoparasites, including Syphacia obvelata, Syphacia muris, Aspiculuris tetraptera, Spironucleus muris, and T. muris, as well as a housekeeping gene in feces. The multiplex PCR could identify an equivalent infection of pinworm, Spironucleus muris and T. muris, with a detection limit of as few as 10 copies. Furthermore, dual infections with up to 100-fold differences and triple infections with 10-fold differences in parasite loads can also be detected. In comparison of traditional methods with the multiplex PCR assay, 76 rodents from 11 research colonies and 3 pet shops and additional 27 fecal samples from laboratory rodents were screened for the infestation status of the five endoparasites. The multiplex PCR had higher sensitivity (97.2–100%) and accuracy (99–100%) than those of the traditional antemortem (sensitivity: 83–100%; accuracy: 94–100%) and postmortem methods (sensitivity: 75–100%; accuracy: 92.1–100%). In addition, an early stage of S. obvelata contamination in a SPF laboratory animal colony was also successfully detected by this multiplex PCR assay. This Pinworm/Spironucleus/Tritrichomonas/Actin Multiplex PCR assay should be a powerful tool to screen endoparasite infestations in laboratory colonies without animal sacrifice.
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