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Ubiquitin-specific protease 18 (USP18) is known as an inhibition factor and has been associated with the innate immune response to pathogens. USP18 is the only deconjugating protease with specificity for interferon-stimulated gene 15 (ISG15), which is supposed to be missing in birds. To analyze the efficacy of goose USP18 (goUSP18) against Tembusu virus (TMUV) infection, we first cloned USP18 homologous cDNA from TMUV infected geese. The coding sequence was 1131 bp, and the deduced amino acid sequence shared conserved motifs with its homologues. Tissue-specific expression has shown that goUSP18 transcripts are strongly expressed in the spleen and liver of adult geese, as well as in the pancreas of goslings. Moreover, the goUSP18 transcripts were induced by goose interferons (goIFN) in goose embryo fibroblasts (GEF) and by TLR ligands in peripheral blood mononuclear cells (PBMC). Notably, goUSP18 transcripts were highly up-regulated by TMUV infection compared to the basal level in uninfected birds. Taken together, these results suggested that goUSP18 was involved in host innate immunity against TMUV infection.

Duck hepatitis A virus (DHAV) is the most common aetiologic agent of duck virus hepatitis (DVH), causing substantial economic losses in the duck industry worldwide. In China, officially approved DHAV-1 live-attenuated vaccines have been used widely to vaccinate breeder ducks since 2013. However, following the reports of DVH outbreaks, it has become necessary to assess the epidemiological situation of this virus in China. We conducted molecular epidemiological analyses of 32 DHAV field isolates while analysing the samples from ducks suspected of having hepatitis collected from commercial duck farms in China between May 2010 and December 2015. Considerable changes were observed in the epidemiology of DHAV-1 and DHAV-3 in China over time. A higher number of DHAV-1 strains were isolated during 2010-2012, coinciding with the widespread use of officially approved DHAV-1 live vaccine strains beginning in 2013. In contrast, a higher rate of DHAV-3 causing DHAV infections was observed between 2013 and 2015. Phylogenetic analyses based on the full-length VP1 gene were performed on these field isolates and using reference strains available in GenBank. DHAV-1 field isolates were evaluated in two groups: one group closely related to prototype strains and circulating in China between 2010 and 2012 and another group exhibiting genetic and serological differences from prototype strains. All DHAV-3 strains isolated in this study were grouped as monophyletic, which has become the predominant viral type, particularly in Shandong and Sichuan provinces, since 2013. In conclusion, these data provide updated information on the genetic and serological diversity of DHAV-1 and DHAV-3, and our findings may serve as a foundation for the prevention of, and vaccine development for, DHAV in China.

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.

The US2 protein has been reported to contribute to duck enteritis virus (DEV) infection; however, its kinetics and localization during infection, and whether it is a component of virion, have not been previously reported. To elucidate the function of DEV US2, US2 was amplified by polymerase chain reaction (PCR) and inserted into pET-32a(+); this was expressed, the recombinant US2 protein was purified, and a polyclonal antibody generated. In addition, the kinetics and localization of the US2 gene and protein were determined by quantitative real-time fluorescent PCR, ganciclovir (GCV), and cycloheximide (CHX) treatment, western-blot, and indirect immunofluorescence assay. The packaging of US2 into DEV virions was revealed by a protease protection assay. US2 was found to be transcribed 24 h post-infection (pi) and peaked at 72 h pi; the US2 protein was detected 48 h pi, except in the presence of GCV or CHX. US2 was packed into virions and also localized to the plasma membrane and cytoplasm in infected cells. The results showed that the DEV US2 is a late gene, and that its encoding protein could be a tegument component localized mainly in the cytoplasm. This study provides useful data for the further analysis of DEV US2, including an explanation for the genetic conservation among alphaherpesviruses.

To investigate tetracycline resistance and resistant genotype in Riemerella anatipestifer, the tetracycline susceptibility of 212 R. anatipestifer isolates from China between 2011 and 2017 was tested. The results showed that 192 of 212 (90.6%) R. anatipestifer isolates exhibited resistance to tetracycline (the MICs ranged from 4 to 256 μg/ml). The results of PCR detection showed that, 170 of 212 (80.2%) R. anatipestifer isolates possessed the tet(X) gene. Other genes, including tet(A), tet(M), tet(Q), tet(O), tet(B), and tet(O/W/32/O), were found at frequencies of 20.8, 4.7, 1.4, 0.9, 0.9, and 0.5%, respectively. However, tet(C), tet(E), tet(G), tet(K), and tet(W) were not detected in any isolate. In these tet gene positive strains, 31 (14.6%), 2 (0.9%), 5 (2.4%), 1 (0.5%), 3 (1.4%) were detected containing tet(A)/tet(X), tet(M)/tet(O), tet(M)/tet(X), tet(O)/tet(X), and tet(Q)/tet(X) simultaneously, respectively. One isolates, R131, unexpectedly contained three tet genes, i.e., tet(M), tet(O), and tet(X). Sequence analysis of the tet gene ORFs cloned from R. anatipestifer isolates confirmed that tet(A), tet(B), tet(M), tet(O), tet(Q) and an unusual mosaic tet gene tet(O/W/32/O) were present in R. anatipestifer. The MIC results of R. anatipestifer ATCC 11845 transconjugants carrying tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes exhibited tetracycline resistance with MIC values ranging from 4 to 64 μg/ml. Additionally, the tet(X) gene could transfer into susceptible strain via natural transformation (transformation frequencies of ~10−6). In conclusion, the tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes were found and conferred tetracycline resistance in R. anatipestifer isolates. Moreover, the tet(X) is the main mechanism of tetracycline resistance in R. anatipestifer isolates. To our knowledge, this is the first report of tet(A), tet(B), tet(M), tet(O), tet(Q), and mosaic gene tet(O/W/32/O) in R. anatipestifer.

In order to study the supporting of deep surrounding rock with zonal disintegration tendency, the zonal disintegration phenomenon of deep surrounding rock under three supporting forms is analyzed by the ABAQUS finite element software in this paper, and three supporting forms are un-supporting, bolting and grouting, and combined “Bolting and grouting plus Anchor rope” supporting. The results show that the different effects to zonal disintegration under different supporting forms will occur. Supporting can help to restrain the zonal disintegration of the reinforcement part advantageously, and also lower rupture degree of zonal disintegration and reduce the size of rupture zone. Meanwhile, the stability of surrounding rock is improved. But zonal disintegration may occur outside reinforcement part under greater ground stress. The results are great importance to a better understanding of the deep roadway supporting.

The OmpA/MotB gene from RA by our lab was sequenced. And the molecular characteristic of this gene was analyzed with bioinformatics software. The result indicated that an open reading frame(ORF) containing 648bp nucleotides was preliminarily identified by aligning with gene bank database by software of BlastN and ORF Finder. The GC content of RA OmpA/MotB gene was 36.88% and encoded a 215 amino acids in this peptide. And from the analysis results we know that this gene sequence didn’t contain a successive at least two rare codons string. Phylogenetic tree of the amino acids sequences showed this gene has not very high similarity with the other 15 OmpA/MotB protein sequence.

Objective: The aim was to analyze Riemerella anatipestifer (RA) Cas1 gene and acquire more useful information for guiding the further study of the gene. Methods: Using bioinformatics method, sequence analysis of RA Cas1 gene was carried out with some databases and software. Results: Composition analysis of RA Cas1 gene, and homology search, sequence alignment, phylogenetic tree analysis and physicochemical property analysis of RA Cas1 protein were performed. Homology search revealed that Cas1 protein sequence of R. anatipestifer showed high similarity to those of strains within the Genus Capnocytophaga and Chryseobacterium, in accordance with the result of multiple sequence alignment and phylogenetic tree analysis. The rare codon analysis showed that there was little rare codon string in RA Cas1 gene. Conclusion: Cas1 gene of R. anatipestifer was evolutionally closely related to those of the Genus Capnocytophaga and Chryseobacterium, and can be over-expressed in E.coli without considering the impact of rare codons on translation.

The analysis on codon usage bias of OmpA/MotB gene of Riemerella anatipestifer (RA) may provide a basis for understanding the evolution and pathogenesis of RA and for selecting appropriate host expression systems to improve the expression of target genes in vivo and in vitro. In our study, a comparative analysis of the codon usage bias in the newly discovered RA OmpA/MotB gene and the OmpA/MotB gene of 20 reference flavobacteriaceae was performed. The results of the codon adaptation indes (CAI), effective number of codon (ENC), and GC3s values indicated that synonymous codon usage bias in the OmpA/MotB gene of flavobacteriaceae. The results showed that codon usage bias in the RA OmpA/MotB gene was strong bias towards the synonymous codons with A and T at the third codon position. A high level of diversity in codon usage bias existed, and the effective number of codons used in a gene plot revealed that the G+C compositional constraint is the main factor that determines the codon usage bias in OmpA/MotB gene of flavobacteriaceae. Comparison of the codon usage in the OmpA/MotB gene of different organisms revealed that there were 31 codons showing distinct codon usage differences between the RA and E. coli, 41 between the RA and humans, but 29 between the RA and yeast. Therefore the yeast expression system may be more suitable for the expression of RA OmpA/MotB gene. These results may improve our understanding of the evolution, pathogenesis and functional studies of RA, as well as contribute significantly to the area of flavobacteriaceae research.

In order to provide a basis for understanding the evolutionary relationship and pathogenesis of Riemerella anatipestifer and selecting a appropriate host expression systems to improve the expression of target gene in vivo and in vitro, we identified the codon bias in the newly confirmed D15 gene of Riemerella anatipestifer ATCC 11845 strain and performed comparative analysis of the codon usage bias between D15 gene in R. anatipestifer and the other 10 referenced Flavobacteriaceaes by a series of online bioinformatics softwares. The results revealed that the synonymous codons with A and T at the third codon position had widely usage in the codon of D15 gene of R. anatipestifer. In addition, there were 70 rare codons in the ORF of the D15 of R. anatipestifer, and 32 codons showing distinct usage differences between R. anatipestifer and E. coli, 30 codons between R. anatipestifer and Homo sapiens, 16 codons between R. anatipestifer and yeast, indicated the yeast expression system may be more suitable for the expression of R. anatipestifer genes. The extent of codon usage bias in the D15 gene in R. anatipestifer was highly correlated with the gene expression level, therefore the results may provide useful information for gene classification and functional studies.

Objective: The aim was to identify the codon usage bias between the newly identified Riemerella anatipestifer (RA) major facilitator superfamily mfs_1 gene (GenBank accession No.CP003388.1) and other reference bacteriums. Methods: A comparative analysis of the codon usage bias of the 18 bacteriums was performed by using the CodonW 1.4 program and CUSP (create a codon usage table) program of EMBOSS (The European Molecular Biology Open Software Suite). Results: The results showed obvious differences of the synonymous codon usage bias in the 18 bacteriums indicated by the Codon Adaptation Index (CAI), effective number of codons (ENc), and the value of G+C content at the 3rd codon position. The codon usage pattern of the major facilitator superfamily mfs_1 gene was phylogenetically conserved and similar to that of the major facilitator superfamily mfs_1 gene of the Elizabethkingia anophelis Ag1 Contig17, with a strong bias towards the codons with A and T at the 3rd codon position. A cluster analysis of codon usage pattern of the RA major facilitator superfamily mfs_1 gene with other reference bacteriums demonstrated that the codon usage bias of the major facilitator superfamily mfs_1 genes of the 18 bacteriums had a very close relation with their gene function. The ENc-plot revealed that the genetic heterogeneity in the RA major facilitator superfamily mfs_1 gene and the 18 reference bacteriums were constrained by G+C content, while gene length exerted relatively weaker influences. In addition, comparisons of the codon preferences in the major facilitator superfamily mfs_1 gene of RA with those of Escherichia coli, yeast and humans revealed that there were 36 codons showing distinct usage differences between the RA and E. coli, and 37 between the RA and humans, but only 28 between the RA and yeast. Therefore, the yeast system may be more suitable for the expression of the RA major facilitator superfamily mfs_1 gene. Conclusion: Together, these results may improve our understanding of the evolution, pathogenesis and functional studies of RA and possibly contribute significantly to the area of other bacteriums.

The OmpA/MotB gene from RA by our lab was sequenced. And the molecular characteristic of this gene was analyzed with bioinformatics software. The results indicated that this gene encodes an estimated 215 protein, contains the conserved domain of the OmpA-like protein. The relative molecular weight of the protein was 23.34525kDa, and there is no signal peptide and transmembrane region of the protein. One cAMP-and cGMP-dependent protein kinase phosphorylation site, six Protein kinase C phosphorylation sites, five Casein kinase II phosphorylation sites, five N-myristoylation sites and antigenic determinants were searched in the protein. Most of the total proteins were located in the Outer membrane and cytoplasmic. Phylogenetic tree of the amino acids sequences showed this gene has a close evolutionary relationship with Elizabethkingia anopheles Ag1 and Weeksella virosa DSM16922, indicating that the RA OmpA/MotB protein may have some similar functions with them. These results provided rational data to elucidate biological function and physiological features of the protein.

To provide some evidence for further research on the biological function of capsular polysaccharide family gene and its endcoding protein of Riemerella anatipestifer.we analyzed the sequence of capsular exopolysaccharide gene and physicochemical properties , structure , function of capsular exopolysaccharide family protein by bioinformatics tools online.We found that capsular exopolysaccharide family gene is an open reading frame with 2373 bp in length and contained a single ORF, which consisted of 732 adenine,443 cytosine,321 guanine,877 thymine and a GC content 32.20%. The NC value of the nucleotides sequence of capsular exopolysaccharide family gene was 44.270. Capsular exopolysaccharide family protein consisted of 790 amino acids and had the following characteristics: numerous highly hydrophilic region, two transmembrane domains, zero signal peptide cleavage site, numerous functional sites. We obtain more biological information about capsular exopolysaccharide family gene and its endcoding protein by bioinformatic analysis, which provide some basic information for further research.

Objective: By analyzing the Cas2gene of Riemerella anatipestifer (RA), the aim was to acquire more useful information as a guide of further study. Methods: Using bioinformatics method, sequence analysis of RA Cas2 gene was performed with some databases and software, including homology search, sequence alignment, phylogenetic tree analysis, composition analysis and physicochemical property analysis. Results: Homology search suggested that Cas2 protein sequence of R. anatipestifer showed high similarity to those of strains within Bergeyella zoohelcum and Capnocytophaga canimorsus, consistent with the result of multiple sequence alignment and phylogenetic tree analysis. The rare codon analysis revealed that there was no rare codon string in RA Cas2 gene. Conclusion: Cas2 gene of R. anatipestifer was evolutionally closely related to those strains of Bergeyella zoohelcum and Capnocytophaga canimorsus, and can be over-expressed in E.coli without considering the impact of rare codons on translation.

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.

To detect Salmonella spp. in suspect infected ducks, a loop-mediated isothermal amplification (LAMP) assay was developed. With the help of this assay, we can detect Salmonella enterica serovar Enteriditis and Salmonella enterica serovar Anatis above 6.0 cfu/test and 4.8 cfu/test, respectively, in pure-culture conditions, even in the existence of 0.01 g of duck liver or spleen homogenates; the detection thresholds were still achieved at 6.0 cfu per test tube. Further experiments of the test strains indicated the high specificity of this LAMP assay. In the detection of clinical specimens, a total of 115 Salmonella suspect infected clinical samples was analyzed by traditional culture method, LAMP, and PCR. The results suggested that 11 samples were positive by LAMP and culture methods; however, only 8 specimens were positive by PCR. In consideration of the high sensitivity and fast scanning speed of our LAMP method in clinical specimens, it was more suitable for field application and epidemiological investigation.

In this report, we conduct study on codon composition and codon usage of DPV glycoprotein B (gB) gene, its homologs constitute the most highly conserved family of herpesvirus glycoproteins and are present in members of each herpesvirus subfamily. Our results show that sixty-one codons (excluding the termination codons) in the polypeptide, a high level of diversity in codon usage bias existed for coding the Ala, Gly, Leu, Pro, Arg, Ser, Thr and Val amino acids. Sixteen codons (each for a amino acid), including GCA (Ala), GAT (Asp), GAA (Glu), GGA (Gly), CAT (His), ATA (Ile), AAA (Lys), CTA (Leu), AAT (Asn), CCA (Pro), CAA (Gln), AGA (Arg), TCT (Ser), ACT (Thr), GTA (Val) and TAT (Tyr) were determined as the translationally optimal codons. The codon preferences of DPV gB gene were compared with those of E. coli, yeast, and H. sapiens, we can speculate that the DPV gB gene may be more efficiently expressed in the E. coli system. In summary, knowledge of codon usage of herpesvirus gB genes provides insights into molecular and species evolution, and also plays important role in furthering some biotechnological applications. These would be fruitful areas for further study.

The analysis of codon usage may improve our understanding of the evolution and pathogenesis of DEV（Duck enteritis virus） and allow reengineering of target gene to improve their expression for gene therapy.In this study,we calculated the codon usage bias in DEV UL55 gene and performed a comparative analysis of synonymous codon usage patterns in other 26 related viruses by EMBOSS CUSP program and Codon W on line.Moreover,statistical methods were used to investigate the correlations of these related parameters. By comparing synonymous codon usage patterns in different viruses,we observed that synonymous codon usage pattern in these virus is virus specific and phylogenetically conserved, with a strong bias towards the codons with A and T at the third codon position. Phylogenetic analysis based on codon usage pattern suggested that DEV UL55 gene was clustered with the avian Alphaherpesvirus but diverged to form a single branch. The Neutrality-plot suggested GC12 and GC3s adopt the same mutation pattern,meanwhile,the ENC-plot revealed that the genetic heterogeneity in UL55 genes is constrained by the G+C content, while translational selection and gene length have no or micro effect on the variations of synonymous codon usage in these virus genes.Furthermore, we compared the codon preferences of DEV with those of E. coli, yeast and Homo sapiens.Data suggested the eukaryotes system such as human system may be more suitable for the expression of DEV UL55 gene in vitro. If the yeast and E. coli expression system are wanted for the expression of DEV UL55 gene ,codon optimization of the DEV UL55 gene may be required.

A 561-bp complete open reading frame of the duck enteritis virus(DEV) UL55 gene (GenBank accession No EU071034) was isolated in our laboratory and amplified by common PCR using a pair of specific primers .PCR product containing this ORF was cloned into the vector of PMD18-T. Four evolutionary analysis approaches were used to construct phylogenetic trees of DEV in relation to herpesviruses based on the nucleotide sequences. Bootstrap was used as statistic method to testify the reliablity of the constructed phylogenetic tree. Phylogenetic trees indicated that DEV and other herpesviruses generated from one ancestor and DEV were grouped into the subfamily Alphaherpesvirinae. In our result, DEV showed a close relationship with the genus Mardivirus, but formed a single branch. Partial genomic organization and phylogenetic analysis in the present study provides evidence that DEV was a member of the subfamily Alphaherpesvirinae and should be assigned as an individual genus or group.

To assess the immunogenicity of an inactivated new type gosling viral enteritis virus (NGVEV) vaccine, we investigated 3 different doses of the inactivated vaccine and the inactivated vaccine in conjunction with 3 different doses of recombinant goose interleukin-2 (rGoIL-2) adjuvant. A virus concentration of 10(5) 50% embryo infective dose/mL was subcutaneously inoculated into adult geese divided into 6 groups. The dynamic changes of the humoral and cellular immunity responses elicited by the vaccines in the adult geese postvaccination (PV) were investigated using ELISA, virus neutralization test, and lymphocyte proliferation assay. The clearance of virus from the intestines of geese (175 d PV) was studied by histopathological examination and indirect immunofluorescence assay after virulent NGVEV challenge. This study showed that the inactivated NGVEV vaccine elicits strong humoral and cellular responses in the vaccinated adult geese. The absorbance values of specific anti-NGVEV antibodies, the neutralization antibody titer, and the lymphocyte proliferation index rapidly increased, peaked at about 28 d PV, progressed to the plateau stage, and then decreased slightly. The rGoIL-2 adjuvant enhanced the immune response, and this adjuvant in conjunction with the inactivated NGVEV vaccine induces a significantly higher specific anti-NGVEV antibody absorbance value, neutralization antibody titer, and lymphocyte proliferation index than the non-adjuvant-inactivated NGVEV vaccine (P < 0.05). The inactivated NGVEV vaccine conferred adequate efficient ability to clear NGVEV in vaccinated geese even in the last phase of the vaccination period (175 d PV). The inactivated NGVEV vaccine (0.5 mL/goose) with 1,000 units of rGoIL-2 adjuvant/goose is the most effective dose, thereby eliciting the strongest humoral and cellular immunity responses and providing the most efficacious clearance of NGVEV in vivo.

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.

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.

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.

New type gosling viral enteritis virus (NGVEV) caused a serious disease in naive juvenile goslings. In the described studies the performance of 2 vaccines was analyzed: a vaccine containing adjuvanted inactivated NGVEV and a vaccine containing adjuvanted inactivated NGVEV and recombinant goose IL-2. Breeder geese were subcutaneously vaccinated at the beginning of the egg production period with the vaccines. Breeder geese sham vaccinated with PBS served as control. The cellular and humoral immune responses of the vaccinated breeder geese, as well as the presence of maternally derived antibody to NGVEV, were investigated by ELISA, virus neutralization test, and lymphocyte proliferation assay, respectively. A significantly higher immunogenicity (P0.05) was induced by the inactivated NGVEV-recombinant goose IL-2 adjuvant vaccine compared with the inactivated NGVEV vaccine. The offspring of the vaccinated birds were challenged with virulent NGVEV (100 50% lethal dose) and the protective efficacy of the vaccines was determined. Furthermore, in a field trial the efficacy of the inactivated NGVEV vaccine was recorded from years 2003 to 2007. No clinical signs or abnormal health status were observed in the vaccinated breeder geese and the progeny. After a single application,80% protection was shown in the progeny of geese vaccinated against NGVEV challenge for approximately 5 mo. The extensive field trials further demonstrated that vaccination of breeder geese with the inactivated NGVEV vaccine could be a safe and efficacious means to control NGVE disease. Moreover, the level of maternally derived NGVEV antibody titer in the egg yolk reflected the level of NGVEV antibodies in the breeder geese, suggesting that the egg yolk could be used to monitor the vaccination efficacy in commercial goose breeder flocks.

The objective of the present study was to develop and apply a streptavidin-alkaline phosphatase labeling system of indirect immunohistochemistry (SP-IHC) to detect antigenic distribution and localization regularity of duck plague virus (DPV) vaccine antigens in paraformaldehyde-fixed paraffin-embedded tissues of experimentally vaccinated ducklings. Male New Zealand rabbits were immunized with purified DPV antigens, which were engaged by a combination of differential centrifugation and sucrose-density gradient ultracentrifugation. The rabbit anti-DPV polyclonal antibodies were purified and used as the primary antibodies. Forty-eight 28-d-old DPV-free Pekin ducklings were subcutaneously inoculated with attenuated DPV vaccine in the immunization group and sterile PBS in the control group. The tissues were collected at sequential time points between 4 h and 18 wk postvaccination (PV) and were prepared for SP-IHC observation. The presence of DPV-specific antigens was first observed in the liver and spleen at 12 h PV; in the bursa of Fabricius, thymus, Harderian gland, esophagus, and intestinal tract at 1 d PV; and in the heart, lung, kidney, pancreas, and brain at 3 d PV. The positive staining reaction could be detected in the vaccinated duckling tissues until 18 wk PV, and no positive staining cells could be observed in the controls. The highest levels of positive staining reaction were found in the liver, spleen, bursa of Fabricius, thymus, and intestinal tract, whereas a few DPV vaccine antigens were distributed in the heart, pancreas, and esophagus. The target cells had a ubiquitous distribution, especially in the mucosal epithelial cells, lamina propria cells, macrophages, hepatocytes, and lymphocytes, which served as the principal sites for antigen localization. These findings demonstrated that SP-IHC was a reliable method for detecting antigenic distribution and localization regularity of DPV vaccine antigens in routine paraffin sections. The present study may be useful for describing proliferation and distribution regularity of DPV vaccine in the vaccinated duckling tissues and enhance further studies and clinical application of attenuated DPV vaccine.

Based on the duck plague virus (DPV) UL35 gene sequence that our laboratory obtained (GenBank accession number EF643558), a pair of primers was designed using Oligo6.0 and primer5.0, then the UL35 gene was amplified from DPV CHv strain genomic DNA and cloned into the pMD18-T to construct a clone plasmid pMD18-T-UL35. After identification of the pMD18-T-UL35 by PCR amplification and restriction digestion, the fragment of the UL35 gene was subcloned into the prokaryotic expression vector pET-32a(+). The resultant recombinant plasmid pET-32a(+)-UL35 was then transformed into E. coli BL21 (DE3) strain and optimally-expressed under the induction of 1.0 mmol/L IPTG at 34 degrees C for 5 hours. SDS-PAGE analysis showed the recombinant protein (VP26) had a molecular weight of about 33KDa and accounted for 32.3% of total bacterial protein by gel scanning. The protein was then purified by Ni(2+)-affinity chromatography and used to immunize rabbit for producing the VP26 anti-serum and its antibody titer was up to 1:32 detected by agar diffusion reaction. After the IgG of the polyclonal antibodies was purified by High-Q anion-exchange chromatography, Western blot analysis indicated that the IgG had specific reaction with the VP26. Moreover, the subcellular localization detection was observed using immunofluorescence technique. The results showed that the specific fluorescences appeared relatively few in nucleus in 2 to 8 hours and increased gradually in 12 to 36 hours and eventually reached to the maximum, which aggregated in the spot region of the nucleus after the duck embryo fibroblast (DEF) were infected by DPV. However, there were only a small amount of specific fluorescences in the cytoplasm in 12 hours and increased with the extension of infection time in 24 to 48 hours. The specific fluorescences finally reached to the maximum in the cytoplasm in 72 hours. The results provided significant data for furthering the study on the function of DPV UL35 gene.

Replication of duck plague virus(DPV) in artificially infected ducks were detected by in situ hybridization (ISH) which employed a 37bp oligonucleotide as probe designed according to DPV DNA sequence in GenBank. The results indicated that DPV DNA was detected in liver, intestine and bursa Fabricius at 4 h, in spleen and esophagus at 6h, in thymus at 12h post infection; DPV DNA in lung and kidney was detected only in dead ducks and no positive signal was detected in muscle, heart, cerebrum and pancreas. DPV DNA was distributed in cell nucleus and cytoplasm. Hepatocytes, sinus endodermal cells and Kuffer's cells were the mainly infected cell types in liver. DPV DNA was mainly detected in epithelium of villi, in lamina propria of intestinal villi of duodenum, in stratum spinosum of esophagus, and in epithelium, cortex, medulla of bursa Fabricius. The positive signals were mainly detected in medulla of thymus, lymphocytes and macrophages of spleen. The research suggests that ISH is a direct and specific method in detecting DPV DNA in paraffin sections and it's also a good method for virus diagnosis and DNA location of DPV.

Kirby-Bauer tests were used to analyze the antibiotic resistance of 224 isolates of Riemerella anatipestifer isolated between 1998 and 2005. Among the 36 antibiotics tested, 50% of the analyzed isolates were resistant to ampicillin, ceftazidime, aztreonam, cefazolin, cefepime, cefuroxime, oxacillin, penicillin G, rifampin, and trimethoprim/sulfamethoxazole. Higher levels of resistance were detected for aztreonam, cefepime, oxacillin, penicillin G, ceftazidime, and trimethoprim/sulfamethoxazole (87.8%, 64.3%, 88.6%, 86.9%, 75.9%, and 79.2% resistance, respectively). The lowest resistance rates were observed for amikacin (9.5%), cefoperazone (7.2%), imipenem (3.2%), and neomycin (9.5%). Four isolates were found to be resistant to 29 different antimicrobials. Riemerella anatipestifer drug resistance profiles changed over time, and the only consistent patterns observed were the resistance of R. anatipestifer to cefoperazone, piperacillin, spectinomycin, and aztreonam. In addition to determining the antibiotic-resistance profiles of R. anatipestifer isolates, we also examine the therapeutic efficacy of these antibiotics against lethal R. anatipestifer infection in ducks in vivo. According to these data, we have extrapolated an antibiotic treatment approach for veterinarians attending flocks of ducks. These data suggest that disk-diffusion analyses can be extrapolated to predict in vivo efficacy, thereby facilitating the identification of effective antibacterial treatments and potentially diminishing the irresponsible use of antibiotics.