During the study period (February to September 2004), our laboratory received 450 sick, moribund, or dead ducks from 25 flocks in the western and central provinces of Thailand. In the detailed studies ( Table 1 ), blood was sampled for serologic analysis by the hemagglutination inhibition (HI) test. All moribund ducks were euthanized, and their internal organs were collected, fixed with 10% buffered formalin, and processed for histopathologic analysis. Additionally, parts of the brain, lung, trachea, intestine, liver, pancreas, kidney, ovary, oviduct, testes, heart, and tight muscle were collected for virus isolation. The tissues were ground and filtered through 0.2−μ filters. The filtrates of each organ were injected into 9- to 11-day-old embryonated chicken eggs and incubated at 37ºC for 2 days. The eggs were observed daily to determine whether death occurred. The allantoic fluid was harvested and tested for influenza virus by HI assay. Any positive sample was then subtyped for H5N1. A second egg passage was performed if the embryonated eggs were still alive 72 hours after injection.
H5N1 Subtyping. Avian influenza virus was subtyped by HI assay by using antiserum specific against the H5 hemagglutinin. Reverse-transcription polymerase chain reaction (RT-PCR) analysis was used for H5 and N1 typing.[14]
Immunohistochemical Testing. To evaluate histologic changes, we used immunohistochemical testing by indirect immunoperoxidase staining as described.[15] Tissue was fixed in formalin before being embedded in paraffin, then cut in 5−μ-thick sections and mounted onto silanized slides.
During the study period (February to September 2004), our laboratory received 450 sick, moribund, or dead ducks from 25 flocks in the western and central provinces of Thailand. In the detailed studies ( Table 1 ), blood was sampled for serologic analysis by the hemagglutination inhibition (HI) test. All moribund ducks were euthanized, and their internal organs were collected, fixed with 10% buffered formalin, and processed for histopathologic analysis. Additionally, parts of the brain, lung, trachea, intestine, liver, pancreas, kidney, ovary, oviduct, testes, heart, and tight muscle were collected for virus isolation. The tissues were ground and filtered through 0.2−μ filters. The filtrates of each organ were injected into 9- to 11-day-old embryonated chicken eggs and incubated at 37ºC for 2 days. The eggs were observed daily to determine whether death occurred. The allantoic fluid was harvested and tested for influenza virus by HI assay. Any positive sample was then subtyped for H5N1. A second egg passage was performed if the embryonated eggs were still alive 72 hours after injection.H5N1 Subtyping. Avian influenza virus was subtyped by HI assay by using antiserum specific against the H5 hemagglutinin. Reverse-transcription polymerase chain reaction (RT-PCR) analysis was used for H5 and N1 typing.[14] Immunohistochemical Testing. To evaluate histologic changes, we used immunohistochemical testing by indirect immunoperoxidase staining as described.[15] Tissue was fixed in formalin before being embedded in paraffin, then cut in 5−μ-thick sections and mounted onto silanized slides.
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