Genomic analysis reveals the population structure and antimicrobial resistance of avian Pasteurella multocida in China.

OBJECTIVES: To investigate the population structure and antimicrobial resistance (AMR) of avian Pasteurella multocida in China. METHODS: Utilizing WGS analysis, we explored the phylogeny using a dataset of 546 genomes, comprising avian P. multocida isolates from China (n = 121), the USA (n = 165), Australia(n = 153), Bangladesh (n = 3) and isolates of other hosts from China (n = 104). We examined the integrative and conjugative element (ICE) structures and the distribution of their components carrying resistance genes, and reconstructed the evolutionary history of A:L1:ST129 (n = 110). RESULTS: The population structure of avian P. multocida in China was dominated by the A:L1:ST129 clone with limited genetic diversity. A:L1:ST129 isolates possessed a broader spectrum of resistance genes at comparatively higher frequencies than those from other hosts and countries. The novel putative ICEs harboured complex resistant clusters that were prevalent in A:L1:ST129. Bayesian analysis predicted that the A:L1:ST129 clone emerged around 1923, and evolved slowly. CONCLUSIONS: A:L1:ST129 appears to possess a host predilection towards avian species in China, posing a potential health threat to other animals. The complex AMR determinants coupled with high frequencies may strengthen the population dominance of A:L1:ST129. The extensive antimicrobial utilization in poultry farming and the mixed rearing practices could have accelerated AMR accumulation in A:L1:ST129. ICEs, together with their resistant clusters, significantly contribute to resistance gene transfer and facilitate the adaptation of A:L1:ST129 to ecological niches. Despite the genetic stability and slow evolution rate, A:L1:ST129 deserves continued monitoring due to its propensity to retain resistance genes, warranting global attention to preclude substantial economic losses.

Whole-genome analysis reveals possible sources of ALV-J infection in an anyi tile-like gray chicken flock.

Avian leukosis virus (ALV) induces multiple tumors in chicken and is still prevalent in a lot of local flocks in China. In this study, we analyzed the ALV infection status in an Anyi tile-like gray chicken flock by DF1-cells isolation, virus identification, and genome sequencing. Results showed a 29% (29/100) ALV positive rate in this flock. Homology analysis based on env genes illustrated that all these stains belong to subgroup J (92-100% identities) and can be further divided into 5 batches, suggesting a higher diversity of ALV-J within the same flock. The whole-genome analysis of representative stains from each batch confirmed the close relationship between these isolated strains with previously reported strains from different regions (Guangxi, Shandong, and Heilongjiang), revealing the enrichment of different strains in Anyi tile-like grey chickens. This study provides the epidemiological data of ALV-J in a special chicken flock and a reference for the further eradication of ALV in China.

WITHDRAWN: Molecular Evolution and Amino Acid Characteristics of Main Antigen Genes of Clinical Duck-Derived H5N6 Subtype Avian Influenza Virus in East China from 2015 to 2019.

This article has been withdrawn at the request of the authors. The Publisher apologizes for any inconvenience this may cause.

[TAN Bayesian network modeling of online learning decision making of dental undergraduates during the COVID-19 pandemic].

PURPOSE: To perceive the dental undergraduate's policy of coping with online learning and their decision-making laws during the COVID-19 pandemic. METHODS: For dental undergraduate students from the 2016 grade to 2018 grade of Lishui University, two prospective questionnaire surveys were conducted before the online course starting and four weeks later. SPSS Modeler18.0 software was used to screen, review, and analyze the data. TAN (tree augmented naive) Bayesian network models were utilized to analyze and predict variables. Indicators like the overall prediction accuracy, receiver operating characteristic curve (ROC curve), and area under the ROC curve(AUC value) were applied to evaluate the model's predicting performances. RESULTS: The case score of each survey was 422 and 382, and the Cronbach's α coefficients of internal consistency were 0.91 and 0.82. Among the decision-making variables in the aspect of "whether to preview online learning materials", the top-two variables were "looking forward to the semester beginning" and "the validity of the network materials". In speaking of "whether the online courses meet the offline course standards", the top-three variables were "the rhythm of lecturing on live or in recorded videos", "how many online tasks', and" the data frame and organization". The overall prediction accuracy of each constructed TAN Bayesian network model was 89.42% and 87.82%, and their AUC values were 0.75 and 0.93, respectively. CONCLUSIONS: To truly make online courses comparable to the off-line curriculum, teachers should fully understand how the students cope with their online learning at first. Then, only by perceiving and recognizing the students' expectations for education, by efficiently preparing and organizing online materials with all-round, clearly-structured, vivid, comprehensible contents and moderate difficult tasks, by well interacting with students through different websites and social media, can we truly achieve " ongoing learning with suspended class".

Network Pharmacology-Based Analysis of the Underlying Mechanism of Hyssopus cuspidatus Boriss. for Antiasthma: A Characteristic Medicinal Material in Xinjiang.

BACKGROUND: Hyssopus cuspidatus Boriss. (Shen Xiang Cao (SXC)), a traditional medicine herb in Xinjiang, has a long history of being used by minorities to treat asthma. However, its active antiasthmatic compounds and underlying mechanism of action are still unknown. The aim of this study was to investigate the bioactive compounds and explore the molecular mechanism of SCX in the treatment of asthma using network pharmacology. METHODS: The compounds of SCX were collected by a literature search, and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and SwissTargetPrediction were used to predict targets and screen active compounds. Moreover, asthma-related targets were obtained based on DisGeNET, Herb, and GeneCards databases, and a protein-protein interaction (PPI) network was built by the STRING database. Furthermore, the topological analysis of the PPI and SXC-compound-target networks were analyzed and established by Cytoscape software. Finally, the RStudio software package was used for carrying out Gene Ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. AutoDock tools and AutoDock Vina were used to molecularly dock the active compounds and key targets. RESULTS: A total of 8 active compounds and 258 potential targets related to SXC were predicted, and PPI network screened out key targets, including IL-6, JUN, TNF, IL10, and CXCL8. GO enrichment analysis involved cell responses to reactive oxygen species, oxidative stress, chemical stress, etc. In addition, KEGG pathway analysis showed that SXC effectively treated asthma through regulation of mitogen-activated protein kinases (MAPK) signaling pathways, interleukin 17 (IL-17) signaling pathways, toll-like receptor (TLR) signaling pathways, and tumor necrosis factor (TNF) signaling pathways. CONCLUSION: The preliminary study that was based on multiple compounds, multiple targets, and multiple pathways provides a scientific basis for further elucidating the molecules involved and the underlying antiasthma-related mechanisms of SXC.

Differential metabolism-associated gene expression of duck pancreatic cells in response to two strains of duck hepatitis A virus type 1.

Several outbreaks of duck hepatitis A virus type 1 (DHAV-1), which were characterized by yellow coloration and hemorrhage in pancreatic tissues, have occurred in China. The causative agent is called pancreatitis-associated DHAV-1. The mechanisms involved in pancreatitis-associated DHAV-1 infection are still unclear. Transcriptome analysis of duck pancreas infected with classical-type DHAV-1 and pancreatitis-associated DHAV-1 was carried out. Deep sequencing with Illumina-Solexa resulted in a total of 53.9 Gb of clean data from the cDNA library of the pancreas, and a total of 29,597 unigenes with an average length of 993.43 bp were generated by de novo sequence assembly. The expression levels of D-3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which are involved in glycine, serine, and threonine metabolism pathways, were significantly downregulated in ducks infected with pancreatitis-associated DHAV-1 compared with those infected with classical-type DHAV-1. These findings provide information regarding differences in expression levels of metabolism-associated genes between ducks infected with pancreatitis-associated DHAV-1 and those infected with classical-type DHAV-1, indicating that intensive metabolism disorders may contribute to the different phenotypes of DHAV-1-infection.

Development of a Colloidal Gold Immunochromatographic Assay for Duck Enteritis Virus Detection Using Monoclonal Antibodies.

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 103 TCID50/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions.

First report of the multiresistance gene cfr in Pasteurella multocida strains of avian origin from China.

OBJECTIVES: The aim of this study was to investigate the presence and genetic environment of the multiresistance gene cfr gene in Pasteurella multocida of avian origin from China. METHODS: A total of 113 P. multocida isolates were collected from sick poultries (ducks, chickens and geese) from 2003 to 2016 in Southern China and were screened for the presence of the cfr gene by PCR. The cfr-carrying P. multocida strains were subjected to antimicrobial susceptibility testing, S1 nuclease PFGE and Southern blot hybridisation, conjugative transfer and analysis of genetic environment of the cfr gene. RESULTS: Among 113 P. multocida isolates, strains FJ6671 and FJ6683 from Muscovy duck harboured the cfr gene and presented a multiresistant phenotype. The cfr gene in the two strains was located on an ∼40-kb conjugative plasmid in different genetic environments, including ISApl12-cfr-IS26 and IS26-cfr-IS256. CONCLUSIONS: These results demonstrate plasmid-carried cfr in P. multocida and suggest that transposition and homologous recombination mediated by IS26, ISApl1 and IS256 might have played an important role in transfer of the cfr gene in P. multocida. To the best of our knowledge, this is the first report of the cfr gene in P. multocida. Active and ongoing surveillance of cfr in P. multocida is urgently warranted.

M-like protein SrM is not crucial to the virulence of a novel isolate of Streptococcus equi subsp. ruminatorum from Macaca mulatta.

In this study, a Streptococcus strainnamed FJ1804, was isolated from a blood sample collected from a dead Macaca mulatta in China and, was subsequently classified as Streptococcus equi subsp. ruminatorum (S.e. ruminatorum) through 16S rRNA gene sequence analysis. After whole genome sequencing and analysis, an M-like protein encoding gene that encodes an SrM protein that is homologous to the crucial S.e. zooepidemicus crucial virulence factor SzP, was identified in the genome of FJ1804. To determinethe function of SrM in this bacterium, a strain deleted of srm as well as a complement strain were constructed. The results of in vitro cell adherence, invasion and phagocytosis assays and in vivo animal challenge and histopathology showed that the anti-phagocytosis was decreased and the adherence rate was increased in the srm deletion strain, whereas the invasion rate, pathological features and LD50 values inboth zebrafish and BALB/c mice model showed no difference compared to that observed for the WT strain. To the best of our knowledge, this is first of an infection caused by S.e. ruminatorum, which is a newly identified zoonotic pathogen, in Macaca mulatta, and our data suggest that, compared with other S.e. zooepidemicus strains, the SzP homologous protein is not crucial to the virulence of this bacterium.

Specific detection and differentiation of classic goose parvovirus and novel goose parvovirus by TaqMan real-time PCR assay, coupled with host specificity.

BACKGROUND: Classic goose parvovirus (cGPV) causes high mortality and morbidity in goslings and Muscovy ducklings. Novel GPV (N-GPV) causes short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks. Both cGPV and N-GPV have relatively strict host specificity, with obvious differences in pathogenicity. Specific detection of cGPV and N-GPV may result in false positives due to high nucleotide similarity with Muscovy duck parvovirus (MDPV). The aim of this study was to develop a highly specific, sensitive, and reliable TaqMan real-time PCR (TaqMan qPCR) assay for facilitating the molecular detection of cGPV and N-GPV. RESULTS: After genetic comparison, the specific conserved region (located on the NS gene) of cGPV and N-GPV was selected for primer and probe design. The selected regions were significantly different from MDPV. Through a series of optimization experiments, the limit of detection was 50.2 copies/µl. The assay was highly specific for the detection of cGPV and N-GPV and no cross-reactivity was observed with E. coli., P.M., R.A., S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV, DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV. The assay was reproducible with an intra-assay and inter-assay variability of less than 2.37%. Combined with host specificity, the developed TaqMan qPCR can be used for cGPV and N-GPV in differential diagnoses. The frequency of cGPV in Muscovy duckling and goslings was determined to be 12 to 44%, while N-GPV frequency in Mule ducks and Cherry Valley ducks was 36 to 56%. Additionally, fluorescence-positive signals can be found in Mule duck embryos and newly hatched Mule ducklings. These findings provide evidence of possible vertical transmission of N-GPV from breeding Mule ducks to ducklings. CONCLUSIONS: We established a quantitative platform for epidemiological investigations and pathogenesis studies of cGPV and N-GPV DNA that was highly sensitive, specific, and reproducible. N-GPV and cGPV infections can be distinguished based on host specificity.

Specific detection of the novel goose astrovirus using a TaqMan real-time RT-PCR technology.

Recently, a novel goose astrovirus (N-GoAstV) was discovered in China, with the transmission route of N-GoAstV unclear. In this study, we developed a TaqMan-based real-time RT-PCR (qRT-PCR) assay for the detection of N-GoAstV infection. After the optimization of the qRT-PCR assay conditions, the results demonstrated that the lower limit of detection for N-GoAstV was 33.4 copies/µL. No cross-reactivity was observed with other goose-origin viruses. Intra-assay and inter-assay variability were ≤1.36% and 2.34%, respectively. N-GoAstV was detected in both field samples, embryos and newly hatched goslings by qRT-PCR assay, provided the view that N-GoAstV may be both horizontally and vertically transmitted. The established qRT-PCR method showed high specificity, sensitivity, and reproducibility, which can be used in future investigations on the pathogenesis and epidemiology of N-GoAstV.

Development and application of a fiber2 protein-based indirect ELISA for detection of duck adenovirus 3.

Duck adenovirus 3 (DAdV-3) is a newly identified duck adenovirus that has recently emerged in China. The incidence of duck infection caused by this virus is very high, with very large economic losses to the poultry industry. Thus, there is an urgent need for a serological assay for the specific detection of DAdV-3. To this end, prokaryotic expression of the fiber2 protein of DAdV-3 was used as a coating antigen to establish an indirect enzyme linked immunosorbent assay (ELISA) method for the specific detection of antibodies against DAdV-3. The method was found to be specific, repeatable and more sensitive than the agarose gel precipitation test (AGP). This indirect ELISA method based on the recombinant fiber2 protein may be used for the clinical detection of DAdV-3 infection and for monitoring antibody levels after vaccine immunization and is of great significance for the effective prevention and control of the disease.

A duplex PCR assay for the simultaneous detection and differentiation of Muscovy duck parvovirus and goose parvovirus.

Both Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) can cause high mortality and morbidity in Muscovy ducklings. MDPVs and GPVs share high nucleotide identity, which can cause errors during differential diagnosis. In this study, the NS genes of both MDPVs and GPVs were chosen for the design of specific primers after genetic comparison. Only three primers (GF1, MF1 and MGR1) were designed for the duplex PCR assay: GF1 is specific for GPV only; MF1 is specific for MDPV only; and MGR1 is highly conserved for both MDPV and GPV. After a series of optimization experiments, the duplex PCR assay amplified a 161-bp fragment specifically for GPV, a 1197-bp fragment specifically for MDPV, and two fragments (161-bp and 1197-bp) for both GPV and MDPV. The lowest detection limit was 103 copies/µl. No amplification was obtained using nucleic acids from other pathogens (including DAdV-A, DuCV, DEV, GHPV, R.A., E. coli., P.M. and S.S.) occurring in Muscovy ducks. Application of the duplex PCR assay in field samples showed that even one-day-old Muscovy ducklings were both MDPV-positive and GPV-positive. In conclusion, a duplex PCR assay for the simultaneous detection and differentiation of MDPV and GPV was established using only three highly specific primers. Our finding suggested that country-wide vaccination with MDPV and GPV vaccines in waterfowls are necessary.

Novel goose parvovirus in domestic Linwu sheldrakes with short beak and dwarfism syndrome, China.

Recently, short beak and dwarfism syndrome (SBDS) had a sudden outbreak in Cherry Valley duck flocks, followed by Pekin ducks and mule ducks in various regions of mainland China. This widely spreading infectious disease was characterized by growth retardation, smaller beak and tarsus with high morbidity and low mortality rate. In this study, we identified and characterized virus from domestic Linwu sheldrakes (namely as HuN18) with SBDS. HuN18 isolates shared high nucleotide identity with novel goose parvovirus (N-GPV). A 5110-nucleotide full-length genome sequence of HuN18 was found with no deletion in ITR region. Alignment studies of HuN18 showed 96.8%-99.0% identity with other N-GPVs and 92.9%-96.3% identity with classic GPV. According to the recombination analysis, HuN18 showed the potential major parent was the N-GPV sdlc01 strain, the potential minor parent was the classical GPV Y strain, and the secondary potential minor parent was the SYG61v strain. To the best of our knowledge, this is the first report of N-GPV in domestic Linwu sheldrakes with SBDS; these data provide evidence that attenuated live viruses are involved in genetic recombination with prevailing wild parvoviruses, which contributes to the novel emerging variants of waterfowl parvoviruses.

Comparative pathogenicity of two subtypes (hepatitis-type and pancreatitis-type) of duck hepatitis A virus type 1 in experimentally infected Muscovy ducklings.

Duck hepatitis A virus type 1 (DHAV-1) causes acute hepatitis with high morbidity and mortality in ducklings of the genera Cairina and Anas and is characterized by ecchymotic haemorrhage and necrosis of the liver surface. Since September 2011, a new subtype of DHAV-1 (named pancreatitis-type DHAV-1) has been isolated. This new subtype is characterized by yellowish or haemorrhagic pancreatitis, but with no significant pathological changes in the liver. To further investigate the difference in pathogenicity between hepatitis-type DHAV-1 and pancreatitis-type DHAV-1, we infected Muscovy ducklings with a hepatitis-type DHAV-1 strain, FZ86, or a pancreatitis-type DHAV-1 strain, MPZJ1206, and then compared the resulting gross lesions, histopathological changes, viral distribution and cellular apoptosis in the liver and pancreas of Muscovy ducklings. The results suggested that FZ86 induced a more efficient viral propagation in the liver than MPZJ1206, and the gross and histopathological lesions were also limited to the liver. However, MPZJ1206 induced more effective viral replication in the pancreas than FZ86. The MPZJ1206-infected Muscovy ducklings showed an obviously yellowed and haemorrhagic pancreas, but with no significant pathological changes in the liver. Furthermore, FZ86 induced notable hepatocyte apoptosis and increased the expression of caspase-3 in the liver, whereas MPZJ1206 caused apoptosis in a large number of acinar epithelial cells and elevated the expression of caspase-3 in the pancreas. Taken together, these results demonstrated that pancreatitis-type DHAV-1 has many new pathogenic features which distinguish it from the hepatitis-type DHAV-1. RESEARCH HIGHLIGHTS Pancreatitis-type DHAV-1 (MPZJ1206) was characterized by pancreatic haemorrhage and yellow discolouration, but with no obvious haemorrhage and necrosis in the liver. Pancreatitis-type DHAV-1 (MPZJ1206) exhibits many new pathogenic features which distinguish it from the hepatitis-type DHAV-1 (FZ86).

Isolation and characterization of duck adenovirus 3 circulating in China.

Recently, infectious disease outbreaks characterized by swelling and hemorrhagic liver and kidneys occurred in Muscovy ducklings in China. Four viruses were isolated and identified as adenoviruses by transmission electron microscopy (TEM) and polymerase chain reaction (PCR). Sequence analysis identified the new isolates as duck adenovirus 3 (DAdV-3), species Duck aviadenovirus B. The pathogenicity of the new isolate DAdV-3 FJGT01 was investigated using challenge experiments. The gross lesions in the animal experiment were similar to the clinical lesions observed in the diseased ducks. TEM examination of liver sample showed that virions accumulated and arranged in crystal lattice formations in the nuclei of hepatocytes. The present study provides new information about the epidemiology and characteristics of duck adenovirus associated with Muscovy ducklings.

Comparative pathogenicity of different subtypes of duck hepatitis A virus in Pekin ducklings.

Duck hepatitis A virus (DHAV) is a major pathogen of viral hepatitis in ducks, which is a fatal and contagious disease of young ducklings. Despite the identification of numerous DHAV strains (e.g. DHAV-3, DHAV-2, DHAV-1 and DHAV-1a), the pathogenic differences among the different subtypes have not been evaluated. The objective of this study was to compare the pathogenic properties of three epidemic strains DHAV-3, DHAV-1, and DHAV-1a in mainland China, in a Pekin duckling infection model. We evaluated the pathogenicity of these different subtypes by investigating clinical signs, macroscopic and microscopic lesions, immunohistochemical examination, and viral RNA detection after experimental inoculation of Pekin ducklings with the three different DHAV strains. There was no significant difference in pathogenicity between DHAV-3 and DHAV-1. Pathogenicity of DHAV-1a differed significantly from that of classical duck hepatitis A (DHAV-3 or DHAV-1), in that there were no clinical signs of opisthotonos. More importantly, pancreatic bleeding or yellowing, and spleen swelling and bleeding were the predominant lesions in the DHAV-1a group, while liver and spleen lesions were the main signs in classical hepatitis (DHAV-1/3). Our findings indicate that there are differences in the pathogenicity of different subtypes of DHAV in ducklings, which may be useful for understanding the biological characteristics of the different subtypes of DHAV in ducks.

Genomic analysis of Sheldrake origin goose hemorrhagic polyomavirus, China.

Goose hemorrhagic polyomavirus (GHPV) is not a naturally occurring infection in geese in China; however, GHPV infection has been identified in Pekin ducks, a domestic duck species. Herein, we investigated the prevalence of GHPV in five domestic duck species (Liancheng white ducks, Putian black ducks, Shan Sheldrake, Shaoxing duck, and Jinyun Sheldrake) in China. We determined that the Jinyun Sheldrake duck species could be infected by GHPV with no clinical signs, whereas no infection was identified in the other four duck species. We sequenced the complete genome of the Jinyun Sheldrake origin GHPV. Genomic data comparison suggested that GHPVs share a conserved genomic structure, regardless of the host (duck or geese) or region (Asia or Europe). Jinyun Sheldrake origin GHPV genomic characterization and epidemiological studies will increase our understanding of potential heterologous reservoirs of GHPV.

Development of a PCR assay for detection and differentiation of Muscovy duck and goose parvoviruses based on NS gene characterization.

Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) have both been found to cause high mortality and morbidity in Muscovy ducklings. Specific detection is often rife with false positives due to high identity at the genomic nucleotide level and antigenic similarity between MDPVs and GPVs. In this study, significantly variable regions were found, via non-structural (NS) comparison, between MDPV and GPV NS genes; however, NS genes were conserved within the MDPV and GPV groups. A polymerase chain reaction (PCR) assay for detecting and differentiating MDPVs and GPVs was developed with more specificity based on the NS gene characterization. The assay detected as low as 103 DNA copies of both the MDPV and GPV strains, along with 549 separate base pairs (bp). No bands of the same size from other duck pathogens, including duck circovirus, duck enteritis virus, egg drop syndrome virus, duck-origin goose hemorrhagic polyomavirus, Escherichia coli, Salmonella, Riemerella anatipestifer and Pasteurella multocida were amplified. This indicates that this method for performing PCR provides a useful and reliable alternative tool for more precise differentiation of MDPV and GPV infection in clinical samples.