Immunohistochemical detection and localization of new type gosling viral enteritis virus in paraformaldehyde-fixed paraffin-embedded tissue

Abstract

To determine the distribution and localization of new type gosling viral enteritis virus (NGVEV) in paraformaldehyde-fixed paraffin-embedded tissues of experimentally infected goslings, for the first time, an immunohistochemical (IHC) staining method was reported. Anti-NGVEV polyclonal serum was obtained from the rabbits immunized with purified NGVEV antigen, which was extracted by caprylic-ammonium sulphate method and purified through High-Q columns anion exchange chromatography. Three-day-old NGVEV-free goslings were orally inoculated with NGVEV-CN strain suspension as infection group and phosphate buffered saline solution (PBS) as control group, respectively. The tissues were collected at sequential time points between 0.5 and 720 h post inoculation (PI), and prepared for IHC staining and ultra-structural observation. The positive immunoreactivity could be readily detected in the lymphoid and gastrointestinal organs of infected goslings as early as 48 h PI, in the liver, kidney, pancreas and myocardium from 72 h, and in the cerebrum and cerebellum from 96 h, while it was hardly detected in the respiratory organs at any time. The positive staining reaction could be detected in NGVEV-infected goslings until 600 h PI, and no positive staining cell could be observed in the controls. The highest levels of viral antigen were found in the bursa of Fabricius (BF), thymus, proventriculus, gizzard and intestine tract, moreover, the liver, kidney, spleen, myocardium and pancreas were intensively and widely stained. The target cells had a ubiquitous distribution, especially included the epithelial cells, endothelial cells, superficial and crypt mucosal cells, glandular cells, fibrocytes, macrophages and lymphocytes, which served as the principal sites for antigen localization. The ultra-structural observation by transmission electron microscope (TEM) further indicated that NGVEV particles could be widely detected in the lymphoid and digestive organs of infected goslings from 72 h PI onwards. This work may be useful not only for offering a possibility of routine diagnosis of NGVE, but also for better understanding of the pathogenesis of the disease.