Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR.

Two pairs of primers were designed to bind conserved genomic regions of goose parvovirus (GPV) and goose astrovirus (GAstV) to establish a simple, sensitive, and highly specific duplex quantitative PCR (qPCR) method to simultaneously detect the two viruses. The duplex qPCR can distinguish GPV (melting point: 82.1 °C) and GAstV (melting point: 79.8 °C) by the peaks of their individual melting curves. Mixed testing with other waterfowl viruses produced no nonspecific peaks. The established standard curves showed good linear relationships (R2 > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 × 101 and 6.58 × 101 copies/µL, respectively. Both intra- and inter-assay coefficients of variation were <2%, indicating that the method has good repeatability. Twenty tissue samples from diseased geese were examined with the duplex qPCR assay and conventional PCR. Duplex qPCR showed positive rates of 25% for GPV and 45% for GAstV, and the positive rate for GPV and GAstV coinfection was 15%, slightly higher than the results for conventional PCR. These results indicated that this duplex qPCR method is highly sensitive, specific, and reproducible, and is suitable for epidemiological studies to effectively control the transmission of GPV and GAstV.

A TaqMan-based quantitative real-time PCR assay for identification of the goose circovirus.

Goose circovirus (GoCV) is a potential immunosuppressive virus that poses a great hazard to the goose industry and has been shown to be widely distributed throughout China. We have established a fast, sensitive and highly specific TaqMan real-time quantitative PCR detection method for this virus. Specific primers and probes were designed against the conserved regions of the genomic GoCV Rep gene. The results showed that the assay was highly specific and sensitive for GoCV and did not cross-react with other non-targeted waterfowl viruses. The established method will be helpful for epidemiological detection and may be effective in the prevention and control of the disease.

Complete genome sequence of goose parvovirus Y strain isolated from Muscovy ducks in China.

A goose parvovirus (GPV) Y strain was isolated from Muscovy ducks in Anhui Province of China. By polymerase chain reaction method, its complete genomic sequence was found to be 5,106 bp in length, consisting of 444-bp inverted terminal repeat, 1,844-bp non-structural protein and 2,199-bp capsid protein (VP) regions. Then its sequence was aligned with the sequences of GPV and Muscovy duck parvovirus published in the GenBank using the neighbor-joining method. The phylogenetic analyses based on the VP3 gene sequences revealed that the GPV Y strain along with those from Taiwan belonged to the subgroup IIb, while other GPV strains from Muscovy ducks belonged to the subgroup Ib and most of other GPV strains isolated in China mainland were clustered in the subgroup IIa. The absence of the deduced 703-705NRT glycosylation site in VP region may explain the host specificity of the GPV Y strain. The complete genomic sequence of the GPV Y strain from Muscovy ducks will help to understand the molecular and evolutionary characteristics of GPV.

Coexistence of antibiotic resistance genes, fecal bacteria, and potential pathogens in anthropogenically impacted water.

Microbial indicators are often used to monitor microbial safety of aquatic environments. However, information regarding the correlation between microbial indicators and ecotoxicological factors such as potential pathogens and antibiotic resistance genes (ARGs) in anthropogenically impacted waters remains highly limited. Here, we investigated the bacterial community composition, potential pathogens, ARGs diversity, ARG hosts, and horizontal gene transfer (HGT) potential in urban river and wastewater samples from Chaohu Lake Basin using 16S rRNA and metagenomic sequencing. The composition of the microbial community and potential pathogens differed significantly in wastewater and river water samples, and the total relative abundance of fecal indicator bacteria was positively correlated with the total relative abundance of potential pathogens (p < 0.001 and Pearson's r = 0.758). Network analysis indicated that partial ARG subtypes such as dfrE, sul2, and PmrE were significantly correlated with indicator bacteria (p < 0.05 and Pearson's r > 0.6). Notably, Klebsiella was the indicator bacteria significantly correlated with 4 potential pathogens and 14 ARG subtypes. ARGs coexisting with mobile gene elements were mainly found in Thauera, Pseudomonas, Escherichia, and Acinetobacter. Next-generation sequencing (NGS) can be used to conduct preliminary surveys of environmental samples to access potential health risks, thereby facilitating water resources management.