Wanpo Zhang

Identification and molecular analysis of the highly pathogenic duck hepatitis virus type 1 in Hubei province of China

Duck hepatitis virus types 1 (DHV-1) JX strain was isolated from infected ducklings with clinical symptoms from Hubei province of China. These isolated strains showed high pathogenicity to both duck embryo and duckling in Duck embryo neutralization assay and animal infection experiment. The complete genome of JX strain was sequenced. Comparative genome analysis with other available strains in GenBank indicated that JX strain shared 94-99% similarity at the nucleotide level and 95-99% at amino acid level with other DHV-1 strains. Sequence results showed that mutations of nucleotide and amino acid were mainly distributed in VP1 genes. Our result implied that the VP1 probably was the major virulent determinant of DHV-1. In addition, 13 DHV-1 strains from different area were analyzed in phylogeny and they can be grouped into four distinct lineages. The new-identified JX strain was grouped into one lineage with A66 and C80 strains, which were also isolated from China.

Identification of differentially expressed genes in the growth plate of broiler chickens with thiram-induced tibial dyschondroplasia

Tibial dyschondroplasia (TD) is characterized by expansion of the proximal growth plates of the tibiotarsus that fail to form bone, lack blood vessels, and contain non-viable cells. Thiram (a carbamate pesticide), when fed to young broiler chicks, induces TD with high regularity and precision. We used this experimental model to understand the cause of the defects associated with TD by selecting and identifying the genes differentially expressed in the TD growth plate of broiler chickens. Broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 96 h to induce TD (thiram-fed). mRNA was purified from the growth plates of control and thiram-fed broilers. Forward and reverse-subtracted cDNA libraries were generated by suppression subtractive hybridization technology. Ten selected genes from cDNA libraries were identified by real-time quantitative polymerase chain reaction. All were differentially expressed in TD growth plates (P<0.05 or P<0.01). The levels of collagen type X (Col X), pro-alpha-1 collagen type I (Col I α1), collagen type IX (Col IX), NADH dehydrogenase (NADH DH), cytochrome C oxidase subunit III (COX III), enolase 1, alpha (ENO1), carbonic anhydrase II (CA2) and heat shock protein 90 (Hsp90) mRNA transcripts were up-regulated, while the expression levels of Matrilin 3 (MATN3) and chondromodulin-I (ChM-I) were down-regulated. Col I and Hsp90 were detected by immunohistochemistry at different stages. Given that these genes are involved in matrix formation, endochondral ossification, developmental regulation, electron transport in the mitochondrial respiratory chain and vascularization, our findings may provide new insights into understanding the pathogenesis of TD.

Cytokine gene expression in the livers of ducklings infected with duck hepatitis virus-1 JX strain

Duck hepatitis virus type 1 (DHV-1) causes a highly contagious disease in ducklings and is often associated with liver necrosis, hemorrhages, and high mortality. In the current study, the expression levels of gene transcripts encoding proinflammatory cytokines and the virus were measured by quantitative reverse-transcription PCR in duck livers after infection with a DHV-1 JX isolate obtained from natural cases in Hubei Province, China. In addition, sera IL-1β, IL-6, and alanine aminotransferase levels were quantified. Liver histopathology was examined following DHV-1 infection. The ducklings died within 1 to 2 d postinfection (d.p.i.) because of typical liver degeneration, hemorrhage, necrosis, and bile-duct epithelial cell proliferation. Transcripts of the cytokines IFN-α, IL-6, TNF-α, and IL-10 decreased by 0.5 d.p.i. and then gradually increased at 1 d.p.i. Similarly, DHV-1 JX 3D gene levels in the liver sharply increased at 1 d.p.i. and then maintained a high level. In contrast, liver TNF-α and IL-1β transcripts showed no increased expression of the cytokine gene postinfection and significantly decreased compared with the expression at 0.25 d.p.i., only the expression of IFN-α transcripts increased 128-fold by 1 d.p.i. Changes in the serum IL-6 level remained relatively stable postinfection and not significantly different compared with that of the control (P > 0.05), whereas serum levels of IL-1β significantly decreased at 0.5 d.p.i. and increased from 1 d.p.i. onwards (P < 0.05). Serum alanine aminotransferase levels significantly increased 2 d.p.i. compared with that of the control group (P < 0.01), which seemed to keep with the number of dead ducks. The cytokines exhibited a biphasic pattern following DHV-1 JX infection. Taken together, the data indicated that duckling liver inflammatory responses were produced following experimental DHV-1 JX infection involving multiple cytokines.

Identification of Riemerella anatipestifer genes differentially expressed in infected duck livers by the selective capture of transcribed sequences technique

Riemerella anatipestifer is the causative agent of duck septicaemia. Determination of R. anatipestifer virulence mechanisms will help us to effectively control this contagious agent. The differentially expressed gene profile of R. anatipestifer in infected duck livers was therefore identified and compared with in vitro cultures by selective capture of transcribed sequences analysis. A total of 48 genes were identified, of which 43 were genes that encode enzymes for amino acid biosynthesis and metabolism, intermediary metabolism, and energy metabolism, or proteins for regulatory adaptive responses, general microbial stress response, transport proteins and secreted proteinases. Five were unknown, novel genes. Eight genes representing the categories were randomly chosen and verified by real-time reverse transcriptase-polymerase chain reaction analysis. All were upregulated by R. anatipestifer in infected duck livers, with changes ranging from 1.44-fold to 4.62-fold compared with in vitro cultures. The results from the present study revealed a gene expression profile of R. anatipestifer in infected duck livers. The unknown but novel genes may be potential novel virulence factors for R. anatipestifer. In conclusion, the data from this study will provide a molecular basis for further study of R. anatipestifer pathogenesis.

Genome Sequence of a Fowl Adenovirus Serotype 4 Strain Lethal to Chickens, Isolated from China

ABSTRACT We report here the complete genome sequence of virulent fowl adenovirus serotype 4 strain HB1510, isolated from a diseased chicken with hydropericardium-hepatitis syndrome in Hubei, China, in 2015. The viral genome is 43,721 bp long, and sequence analysis showed an 11-amino-acid deletion in open reading frame 29 (ORF29).

Apoptosis induction in duck tissues during duck hepatitis A virus type 1 infection

To investigate the role of apoptosis in duck viral hepatitis pathogenesis, 4- and 21-d-old ducks were inoculated with duck hepatitis A virus serotype 1 and killed at 2, 6, 12, 24, and 48 h postinfection. TdT-mediated dUTP nick-end labeling was used to detect apoptosis cells. Expression profiles of apoptosis-related genes including caspase-3, -8, -9, and Bcl-2 in spleen, bursa of Fabricius, liver, and the quantity of virus in blood were examined using real-time PCR. The TdT-mediated dUTP nick-end labeling analysis indicated there was a significant difference of apoptotic cells between treatments and controls. The same difference also appeared in virus amount variation in blood during infection. Gene expression analysis revealed that the apoptosis-related gene expression profile was different in the 2 groups, and also different between various organs. This study suggested that apoptosis may play an important role in duck hepatitis A virus serotype 1 infection, and apoptosis suppression might facilitate virus multiplication, resulting in the highest virus concentration in the host.

Isolation and characterization of Streptococcus gallolyticus subsp. pasteurianus causing meningitis in ducklings

Increased mortality was observed in a group of 10-day-old ducklings in China in 2010. The dead ducklings were characterized by meningitis, suggesting microbial infection as the cause of disease. Laboratory investigation led to the isolation of a bacterial strain designated as AL101002 from the brain of dead ducklings. Subsequent experimental infections with this strain resulted in marked symptoms in the ducklings similar to those observed in nature outbreaks. Gram-staining revealed Gram-positive cocci in pairs or short chains. Phenotypical analysis revealed the microorganism as Streptococcus pasteurianus. DNA sequencing revealed that the isolate was Streptococcus gallolyticus subsp. pasteurianus. Experimental infection with AL101002 resulted in the death of ducklings with meningitis. To the best of our knowledge, this is the first report of the isolation of S. gallolyticus subsp. pasteurianus with high virulence in ducklings.

Evaluation of a thermostable Newcastle disease virus strain TS09-C as an in-ovo vaccine for chickens

In-ovo vaccination is an attractive immunization approach for poultry industry. However, most of the Newcastle disease virus (NDV) vaccine strains used after hatch are unsafe, as in-ovo vaccines, due to their high pathogenicity for chicken embryos. In this study, we evaluated the safety and immunogenicity of a thermostable NDV strain TS09-C, derived from V4 strain, as in-ovo vaccine. Chickens in-ovo vaccinated with the parental V4 strain displayed greatly reduced hatchability and severe histopathological lesions in both trachea and intestine tissues, while the hatchability was not affected by in-ovo vaccination withTS09-C strain. The safe dose that infected all chicken embryos without obviously histopathological lesions was 103.0 EID50 per bird. In-ovo vaccination of chickens with TS09-C virus conferred complete protection against virulent NDV challenge. Results suggest that the thermostable NDV strain TS09-C is a safe and immunogenic in-ovo vaccine candidate that can be delivered quickly and uniformly, and induce earlier immune response.

Protein Expressions and Their Immunogenicity from Riemerella anatipestifer Cultured in Iron Restriction Medium

Riemerella anatipestifer was cultured in both iron restriction media and normal media. Two-dimensional gel electrophoresis identified 23 proteins that significantly increased in the iron restriction media. Of them 12 proteins were analyzed with mass spectrography. Nine of 12 proteins belong to 6 different protein families: fibronectin type iii domain protein, secreted subtilase family protein, phosphoglycerate kinase, translation elongation factor, leucine-rich repeat-containing protein, and Galactose-binding domain-like protein. Other 3 proteins were novel with unknown function. Two novel proteins (Riean_1750 and Riean_1752) were expressed in prokaryotic expression systems. The specificities of these 2 novel proteins to R. anatipestifer were confirmed by western-blotting analysis. The ducks immunized with either protein had low mortality challenged by R. anatipestifer, 33.3% and 16.7%, respectively. The ducks developed 100% immunity when immunized with combined Riean_1750 and Riean_1752 proteins. The data suggested 2 novel proteins play important roles in the bacterial survival in the iron restricted environment. They could be used as subunit vaccines of R. anatipestifer.

Necroptosis of Splenic Macrophages Induced by Streptococcus gallolyticus subsp. pasteurianus

SUMMARY A previous study demonstrated that a highly virulent strain of Streptococcus gallolyticus subsp. pasteurianus, designated as the AL101002 strain, induced high mortality in ducklings with splenic lesions. In this study, 42 ducklings were subcutaneously inoculated with the AL101002 strain to study changes in splenic lesions over time. The spleens from these ducklings were significantly enlarged by congestion and edema, and/or showed multiple marbled areas 14 days postinoculation (dpi). The AL101002 strain was reisolated from the spleens and blood and confirmed by immunohistochemistry (IHC) with the use of anti-AL101002 antibody. Histopathologically, the main lesion was macrophage necrosis in the spleens from 1 to 7 dpi. Terminal dUTP nick-end labeling assay, transmission electron microscopy, and IHC by anti-macrosialin antibody (CD68) demonstrated that macrophage necrosis was necroptosis, which was further confirmed by quantitative (real-time) reverse-transcriptase PCR analysis. Two major factors of apoptosis, caspase 3 and caspase 8, did not significantly change during the AL101002 infection, suggesting that apoptosis signals were not activated. However, the key factor mixed lineage kinase like was increased significantly (P < 0.05) from Day 1 to Day 14 dpi. Inflammatory cytokine interleukin-1β and interleukin-6 had significantly (P < 0.01) upregulated expression in the spleens on Day 1 dpi. Tumor necrosis factor α was downregulated from Day 1 to Day 5 dpi, but increased from Day 7 to Day 14. Our results demonstrated that AL101002 strain mainly infects macrophages and resulted in macrophage necroptosis and suggested that macrophage necroptosis in spleens is involved in the pathogenesis of S. gallolyticus subsp. pasteurianus infection in ducklings.