Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV.

The UL13 protein of the duck enteritis virus (DEV), predicted to encode a Ser/Thr protein kinase, belongs to the family of conserved herpesvirus protein kinases (CHPK), which plays an important role in herpesvirus proliferation. In this study, truncated UL13 was expressed as a fusion protein of approximately 44 kDa using a prokaryotic expression system, and this protein was used to generate a specific anti-UL13 antibody. This antibody detected UL13 starting at 4 h post infection in duck embryonic fibroblast cells and identified UL13 to be present in both the cytoplasm and the nucleus. UL13 RNA was found to be transcribed starting at 2 h post infection, and the synthesis of the UL13 mRNA was found to be sensitive to the protein synthesis inhibitor cycloheximide (CHX) and tolerant of the DNA polymerase inhibitor ganciclovir (GCV). Its nuclear location and status as an early gene suggested that DEV UL13 might play important roles in DEV replication, which was confirmed by comparing the proliferation of a UL13-knockout mutant virus, a revertant virus, and the parent virus in cell culture. The specific mechanisms of UL13 in viral replication need to be further studied.

A pair of PCR primers was designed and synthesized to amplify a gyrB gene sequence from Riemerella anatipestifer (RA). A fragment of 194 bp was detected in RA-positive isolates, whereas other isolates were negative, which confirmed the high specificity of the primers and PCR conditions. As little as 1.6 × 10(4) cfu/mL of cultural liquid was required by this method. We compared a 16S rRNA sequence-based PCR method and a Biolog bacterial identification system used in the detection and identification of suspicious isolates of RA in clinical tests. The results showed that the gyrB-based PCR was consistent with the results of the Biolog identification system and was more specific. By applying the gyrB-PCR to detect RA strains in 56 duck livers, a positive rate of 46% (26/56) was observed, whereas the positive rate of 85 throat swabs from clinically healthy ducks was 11%. Thus, this method could be used for the epidemiological investigation and preliminary isolate identification of RA.

Here, we investigated adhesion and invasion of Riemerella anatipestifer (RA) to primary duck embryo fibroblast (DEF) cells. The ability of RA to adhere to, and more importantly, to invade DEF cells was demonstrated by using a gentamicin invasion assay and was confirmed by transmission electron microscopy (TEM). Adhesion of RA could be found by TEM after 1 h of inoculation. Both apoptosis and necrocytosis of DEF were indicated by TEM after 10 h of incubation, which suggested a complex mechanism of DEF cell death induced by RA. Our results showed that internalized RA had the ability to leave the DEF cells. Inhibition studies indicated that RA proteins play a role in adhesion. Moreover, invasion of RA to DEF cells was shown to require rearrangement of actin microfilaments and microtubular cytoskeletal elements. Because the adhesion and invasion ability of RA to DEF cells could be demonstrated in vitro, similar processes might occur in vivo, where DEF cells play a crucial role in the diffusion of RA in ducks.

Here, for the first time, to colocalize new type gosling viral enteritis virus (NGVEV) with histological lesions and in situ apoptosis in the digestive organs (esophagus, proventriculus, gizzard, small intestine, cecum, rectum, liver, and pancreas) and the lymphoid organs (bursa of Fabricius, thymus, Harderian gland, and spleen) of experimentally infected goslings, portions of tissues were collected at sequential infection time points and examined by histopathology for histological lesions, immunohistochemical staining for viral antigens, ultrastructural observation by transmission electron microscope (TEM) for virus particles and apoptotic cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay for in situ apoptosis. The hyperemia, hemorrhage, infiltration of lymphocytes, progressive lymphoid depletion, apoptosis, and necrosis were readily observed in the lymphoid organs and intestine tract by histopathological examination. The NGVEV particles and viral antigens widely appeared in the small intestine and bursa of Fabricius as early as 2 d postinfection (PI) by TEM and immunohistochemical staining, and the presence and quantity of it reached a maximum during 6 to 12 d PI. The principal sites for NGVEV were endothelial cells, epithelia, mucosal cells, glandular cells, fibrocytes, macrophages, and lymphocytes. A series of apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, and formation of apoptotic body were observed by TEM, and the number of apoptotic cells was largely increased from 4 d PI and peaked at 9 d PI by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling analysis. The histological organ lesions and apoptosis in vivo were generally associated with sites of NGVEV localization, which can be regarded as the cause of death. This work may shed light on the pathogenesis of new type gosling viral enteritis and put new insight into the pathogenesis of goose adenovirus.