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.

Numerical and Field Measurement Investigation on Foundation Pit Excavation Blasting of Anchor in Suspension Bridge.

The foundation pit of a suspension bridge project in the Three Gorges Reservoir area is investigated in this paper. The pit is located under an unstable rock mass and landslide body; its base lithology is mudstone. The bridge foundation pit project mainly adopts blasting excavation to accelerate construction progress. However, as a hazardous technique to engineering safety, the explosion vibration easily causes deterioration of the surrounding strata, thereby inducing slope instability and rock mass collapse. Besides, three major challenges should be considered: complex terrain conditions, difficulties in the blasting excavation of anchors, and the extremely high risk of construction. Therefore, comprehensive risk control measures using the methods of hierarchical excavation and minimum charge blasting are put forward. After the measures were verified to be feasible through finite element simulation, it was successfully applied to actual construction. In addition, this paper proposes using fiber concrete to reinforce slope retaining walls, and simulates the reinforced effect based on the research above. The results indicate that the risk control scheme is reasonable, which not only ensures the construction process but also guarantees the stability of the slope and unstable rock body. At the same time, the slope is reinforced with fiber concrete, which effectively decreases the protection wall thickness. Finally, the article can provide a valuable reference for similar engineering projects around the world.

Isolation and Functional Analysis of VvWRKY28, a Vitis vinifera WRKY Transcription Factor Gene, with Functions in Tolerance to Cold and Salt Stress in Transgenic Arabidopsis thaliana.

The grape (Vitis vinifera L.) not only has a long history of cultivation, but also has rich nutritional value and high economic value. However, grapes often face many threats in the growth process. For example, low temperature and salt stress restrict the growth status, yield, and geographical distribution of grapes. WRKY, as one of the largest transcription factor (TF) families in plants, participates in the response of plants to stress. VvWRKY28, a new zinc finger type transcriptional regulator gene, was isolated from Beichun (V. vinifera × V.amurensis) in this study. From the subcellular localization results, it can be concluded that VvWRKY28 was localized in the nucleus. The expression of VvWRKY28 was enriched in leaves (young and mature leaves), and cold and high salt conditions can induce high expression of VvWRKY28. After being transferred into Arabidopsis, VvWRKY28 greatly improved the tolerance of Arabidopsis to low temperature and high salt and also changed many physiological and biochemical indicators of transgenic Arabidopsis to cope with cold and high salt stimulation. The content of malondialdehyde (MDA) was decreased, but for chlorophyll and proline, their content increased, and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were improved. In addition, under cold stress, binding with cis-acting elements promotes the expression of downstream genes related to cold stress (RAB18, COR15A, ERD10, PIF4, COR47, and ICS1). Moreover, it also plays an active role in regulating the expression of genes related to salt stress (NCED3, SnRK2.4, CAT2, SOD1, SOS2, and P5CS1) under salt stress. Therefore, these results provide evidence that VvWRKY28 may play a role in the process of plant cold and salt stress tolerance.

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.

[Analysis of genetic variant in a fetus featuring pontocerebellar hypoplasia type 6].

OBJECTIVE: To explore the genetic basis for a fetus with cerebellar dysplasia and widened lateral ventricles. METHODS: The couple have elected induced abortion after careful counseling. Skin tissue sample from the abortus and peripheral venous blood samples from both parents were collected for the extraction of genomic DNA, which was then subjected to whole exome sequencing. Candidate variant was verified by Sanger sequencing. RESULTS: Prenatal ultrasonography showed increased nuchal translucency (0.4 cm) and widened lateral ventricles. Magnetic resonance imaging revealed infratentorial brain dysplasia. By DNA sequencing, the fetus was found to carry compound heterozygous variants c.1A>G and c.1564G>A of the RARS2 gene, which were inherited from its father and mother, respectively. Among these, c.1A>G was known to be pathogenic, but the pathogenicity of c.1564G>A was unreported previously. Based on the American College of Medical Genetics and Genomics guidelines, the c.1564G>A variant of RARS2 gene was predicted to be likely pathogenic(PM2+PM3+PP3+PP4). CONCLUSION: The compound heterozygous variants c.1A>G and c.1564G>A of RARS2 gene contributed to the fetus suffering from pontocerebellar hypoplasia type 6, which expanded variant spectrum of RARS2 gene.

The association between consumption of monounsaturated fats from animal- v. plant-based foods and the risk of type 2 diabetes: a prospective nationwide cohort study.

Although higher dietary intake of MUFA has been shown to improve glycaemic control and lipid profiles, whether MUFA consumption from different sources is linked to the development of type 2 diabetes (T2D) remains unclear. We aimed to prospectively assess the associations of plant-derived MUFA (P-MUFA) and animal-derived MUFA (A-MUFA) intakes with T2D risk in a nationwide oriental cohort. Overall, 15 022 Chinese adults, aged ≥20 years, from the China Health and Nutrition Survey (CHNS 1997-2011) were prospectively followed up for a median of 14 years. Consumption of MUFA from plant and animal sources was assessed using 3-d 24-h recalls in each survey, and the cumulative average of intake was calculated. Multivariable-adjusted Cox models were constructed to estimate the hazard ratios (HR) of T2D according to quartiles of MUFA intake. P-MUFA were mainly consumed from cooked vegetable oils, fried bread sticks and rice, while A-MUFA were mainly consumed from pork, lard and eggs. Intake of P-MUFA was associated with a higher risk of T2D (HRQ4 v. Q1 1·50 (95 % CI 1·18, 1·90); Ptrend = 0·0013), whereas A-MUFA showed no significant association (HRQ4 v. Q1 0·84 (95 % CI 0·59, 1·20); Ptrend = 0·30). When further considering the cooking method of food sources, consumption of P-MUFA from fried foods was positively associated with T2D risk (HRQ4 v. Q1 1·60 (95 % CI 1·26, 2·02); Ptrend = 0·0006), whereas non-fried P-MUFA were not associated. Intake of MUFA from fried plant-based foods may elevate T2D risk among the Chinese population.

[Identification of a Tp63 gene variant in an abortus with Ectrodactyly, Ectodermal dysplasia, Cleft lip/palate syndrome by whole-exome sequencing].

OBJECTIVE: To detect potential variant in a male fetus suspected for Ectrodactyly, Ectodermal dysplasia, Cleft lip/palate (EEC) syndrome. METHODS: Peripheral blood samples of the fetus and his parents were collected for the extraction of DNA. Whole-exome sequencing was carried out to detect potential variants. Suspected variants were verified by Sanger sequencing. RESULTS: The fetus was found to carry a heterozygous c.673C>T missense variant of the Tp63 gene, which was known to underlie split-hand/split-foot malformation. The same variant was not found in either parents. CONCLUSION: The heterozygous c.673C>T missense variant of the Tp63 gene probably underlies the EEC syndrome in the fetus. Above finding also expanded the phenotypic spectrum for this variant.

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.

Saturated Fatty Acid Intake Is Associated with Total Mortality in a Nationwide Cohort Study.

Background: Dietary intakes of total and specific types of saturated fatty acids (SFAs) in relation to total mortality remain largely unknown in China. Objective: We assessed the associations of total and individual SFA intakes with total mortality in a Chinese nationwide population. Methods: This prospective analysis included 7888 women and 6495 men, aged >20 y, from the China Health and Nutrition Survey (1989-2011). Cumulative mean of SFA intake was calculated based on 3 d of 24-h records in each round of the survey. Multivariable Cox proportional hazard models were used to estimate HR and 95% CI. Results: There were 1011 deaths during a median of 14 y of follow-up. In isocaloric nutrient density models that replace total carbohydrates, intakes of total SFAs and even-chain SFAs were associated with higher total mortality in women but not in men. Compared with the lowest quartiles, the HRs (95% CIs) in the highest quartiles were 1.65 (1.03, 2.62) for total SFAs (P-trend = 0.042) and 1.83 (1.13, 2.94) for even-chain SFAs (P-trend = 0.016) in women. Intake of odd-chain SFAs was associated with lower total mortality both in men [HR (95% CI): 0.64 (0.47, 0.87); P-trend = 0.01] and in women [HR (95% CI): 0.59 (0.42, 0.84); P-trend = 0.0013]. Moreover, intake of medium-chain SFAs was linked with lower total mortality in men [HR (95% CI): 0.64 (0.44, 0.93); P-trend = 0.013]. However, isocalorically replacing 1% of energy from unsaturated fatty acids with even-chain SFAs was associated with higher total mortality in men [HRs (95% CIs): 1.08 (0.97, 1.20) and 1.39 (1.20, 1.60) for replacing PUFAs and MUFAs, respectively]. Conclusions: Total SFA and even-chain SFA intake was associated with higher total mortality in women, whereas intake of odd-chain SFAs was related to lower total mortality in both sexes. This analysis of cohort was registered at clinicaltrials.gov as NCT03281512.

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.

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.

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.

The effects of compression stockings on the energetics and biomechanics during walking.

PURPOSE: The purpose of this study was to explore how compression stockings affect the energetics and biomechanics during walking. METHODS: Sixteen male adults participated in this study. Participants completed walking trials on the treadmill and force plates, wearing compression stockings (CS) or nothing as a control condition (CON). The data obtained included metabolic rate, muscle activation, step frequency and step length as well as their variability, joint kinematics and joint kinetics. RESULTS: The effect of compression stockings on metabolic rate was trivial (CS: 3.81 ± 0.44 W kg-1, CON: 3.83 ± 0.46 W kg-1, p = 0.84, d = 0.05). Activation of calf muscles, step frequency and step length as well as their variability, joint range of motion and joint powers did not show a significant difference between conditions (p = 0.09-0.90, d = 0.01-0.34). The peak knee extension moment during the early stance phase had a tendency to increase (CS: 0.57 ± 0.27 N m kg-1, CON: 0.51 ± 0.28 N m kg-1, p = 0.05, d = 0.19) while the peak knee flexion moment during the late swing phase had a tendency to decrease (CS: 0.16 ± 0.10 N m kg-1, CON: 0.19 ± 0.12 N m kg-1, p = 0.10, d = 0.21). The peak ankle dorsiflexion moment during the early stance phase significantly increased (CS: 0.11 ± 0.06 N m kg-1, CON: 0.08 ± 0.05 N m kg-1, p = 0.02, d = 0.58) while the peak ankle plantar flexion moment during the late swing phase significantly decreased (CS: 1.41 ± 0.12 N m kg-1, CON: 1.47 ± 0.14 N m kg-1, p = 0.02, d = 0.45). CONCLUSIONS: Compression stockings have a limited effect on improving energetics of walking, but they may play a role in improving biomechanics by altering the relative contribution of knee and ankle moments to propulsion.

Development of a TaqMan-based real-time PCR assay for the rapid and specific detection of pigeon torque teno virus.

Pigeon torque teno virus (PTTV), a recently discovered circular DNA virus. Here, we developed a TaqMan-based real-time PCR for rapid and specific detection of PTTV infections with sensitivity up to 49.3 copies/µl. Positive signals can be observed by the assay in pigeon embryonated eggs, which indicted that PTTV can be transmitted vertically. Our findings play important implications for a better understanding the transmission of torque teno virus in pigeons.

Rapid detection of goose hemorrhagic polyomavirus using TaqMan quantitative real-time PCR.

Due to low doses of infection, an efficient and sensitive virus detection method is necessary to detect low amounts of goose hemorrhagic polyomavirus (GHPV). In this study, we have developed a TaqMan real-time PCR (qPCR) specific assay for the detection of GHPV. Specificity assay showed no cross-reactions with other common waterfowl viruses. The standard curve had a linear correlation of 0.997 and efficiency of 99% between the cycle threshold value and the logarithm of the plasmids copy number. The possible lowest detectable concentration was 35.4 copies/µl; 100 times more sensitive than conventional PCR (detection limit, 3.54 × 103 copies/µl). Domestic Jinyun Sheldrakes ducks and their embryonated eggs were found positive of GHPV infection which provides evidence of possible vertical transmission of GHPV.

Specific detection of Muscovy duck parvovirus infection by TaqMan-based real-time PCR assay.

BACKGROUND: Muscovy duck parvovirus (MDPV) causes high mortality and morbidity in Muscovy ducks, with the pathogenesis of the virus still unknown in many respects. Specific MDPV detection is often rife with false positive results because of high identity at the genomic nucleotide level and antigenic similarity with goose parvovirus (GPV). The objective of this study was to develop a sensitive, highly specific, and repeatable TaqMan-based real-time PCR (qPCR) assay for facilitating the molecular detection of MDPV. RESULTS: The specific primers and probe were designed based on the conserved regions within MDPVs, but there was a variation in GPVs of the nonstructural (NS) genes after genetic comparison. After the optimization of qPCR conditions, the detection limit of this qPCR assay was 29.7 copies/µl. The assay was highly specific for the detection of MDPV, and no cross-reactivity was observed with other non-targeted duck-derived pathogens. Intra- and inter-assay variability was less than 2.21%, means a high degree of repeatability. The diagnostic applicability of the qPCR assay was proven that MDPV-positive can be found in cloacal swabs samples, Muscovy duck embryos and newly hatched Muscovy ducklings. CONCLUSIONS: Our data provided incidents that MDPV could be possible vertically transmitted from breeder Muscovy ducks to Muscovy ducklings. The developed qPCR assay in the study could be a reliable and specific tool for epidemiological surveillance and pathogenesis studies of MDPV.

[Analysis of PHEX gene mutations in three pedigrees affected with hypophosphatemic rickets].

OBJECTIVE: To explore the molecular basis for three pedigrees affected with hypophosphatemia vitamin D resistant rickets (X-linked hypophosphatemia, XLH). METHODS: Peripheral blood samples from the three pedigrees were collected. Following DNA extraction, the 11 exons and flanking regions of the PHEX gene were subjected to PCR amplification and direct sequencing. Pathogenicity of identified mutations was evaluated through genotype-phenotype correlation. RESULTS: For pedigrees 1 and 2, pathogenic mutations were respectively identified in exon 8 (c.871C>T, p.R291X) and exon 15 (c.1601C>T, p.P534L) of the PHEX gene. For pedigree 3, a novel mutation (c.1234delA, p.S412Vfs*12) was found in exon 11 of the PHEX gene, which caused shift the reading frame and premature termination of protein translation. CONCLUSION: The three mutations probably account for the XLH in the affected pedigrees. The discovery of novel mutations has enriched the spectrum of PHEX gene mutations.

[Analysis of clinical manifestation and genetic mutations in two patients with Cornelia de Lange syndrome].

OBJECTIVE: To detect potential mutations in two neonates suspected for Cornelia de Lange syndrome (CdLS). METHODS: Peripheral blood samples from the neonates and their parents were collected and analyzed for CdLS-related genes using targeted sequence capture and next-generation sequencing. Suspected mutations were confirmed by direct Sanger sequencing. RESULTS: The neonates were found to respectively carry mutations c.7219C to T and p.D2339Lfs*4 of the NIPBL gene, among which the p.D2339Lfs*4 mutation has not been reported previously. No pathogenic mutation was found in other CdLS-related genes including NIPBL, SMC1A, SMC3, RAD21 and HDAC8. CONCLUSION: The c.7219C to T and p.D2339Lfs*4 mutations of the NIPBL gene probably account for the disease in both patients.