Epidemiological investigations and locally determined genotype diversity of Mycoplasma synoviae in Central China from 2017 to 2019.

Mycoplasma synoviae (M. synoviae) has been identified worldwide to cause respiratory diseases, infectious synovitis, airsacculitis, and eggshell apex abnormalities (EAA) in commercial chickens, which results in substantial economic losses to the poultry industry. Therefore, in this study, 258 flocks were investigated between 2017 and 2019 for M. synoviae by screening samples from Central China. Subsequently, 129 M. synoviae strains were isolated, with a positive rate of 50%. Moreover, a higher incidence of M. Synoviae infections was in layers (74.1%) than in broilers (20%) in this study. The 5'-end conserved segment of the variable lipoprotein hemagglutinin A (vlhA) gene of these isolates was then cloned and sequenced because it is a common genomic target identified so far for M. synoviae genotyping. Genotyping of all isolates was based on the phylogenetic analysis and length analysis of the proline-rich-repeat (PRR) regions, respectively. Phylogenetic analysis based on 5'-end conserved segment of the vlhA gene (76-421 nt) assigned the majority of the occurring strains as being from group 6, and others from groups 2 and 3. Results identified that these isolates were of 6 types: A (38aa), D (23aa), E (19aa), I (28aa), J (20aa), and L (35aa), based on the size of the PRR region analysis. Furthermore, most of the isolates (81.4% were identified as type L. Additionally, the epidemic types included only I and L in 2017; however, the types rose to 5 (A, D, E, I, L) in 2018 and rose to 6 (A, D, E, I, J, L) in 2019. These data showed the genotype diversity of M. synoviae in Central China. The high rate of positive flocks suggests the urgent need to take real-time supervisory controls of this Mycoplasma species in avian flocks.

Rapid and visual detection of novel astroviruses causing fatal gout in goslings using one-step reverse transcription loop-mediated isothermal amplification.

To visually and rapidly detect a novel goose astrovirus (N-GoAstV) causing fatal gout in goslings, an isothermal detection method based on one-step reverse transcription loop-mediated isothermal amplification (one-step RT-LAMP) was established. The one-step RT-LAMP assay for N-GoAstV detection, using Bst 3.0 DNA polymerase with strong reverse transcription activity and primer sets targeting the opening reading frame 1b (ORF1b) of N-GoAstV, could be completed in 30 min using a water bath at 61°C; the detection results could be visually observed by adding a pH-sensitive dye containing phenol red and cresol red. The detection limit of the one-step RT-LAMP assay was 57.8 copies, which was similar to that of reverse transcription-quantitative polymerase chain reaction. The assay specifically detected N-GoAstV without any cross-reaction with other reference viruses, and this was further confirmed using enzyme digestion. These results indicated that the newly established RT-LAMP assay could accomplish reverse transcription, amplification, and visual result determination in one step, and the results obtained via this rapid and cost-effective method could be used to support disease control on farms in terms of N-GoAstV infection.

Novel genotype definition and genome characteristics of duck circovirus in central and Eastern China.

To explore genetic variations in duck circovirus (DuCV) and the molecular epidemiology of its infection, tissue samples were collected from 219 dead ducks from 20 farms in the central and eastern regions of China. All farms tested positive for DuCV, with duck-origin goose parvovirus, reovirus and Tembusu virus having co-infection rates of 100%, 0% and 0%, respectively. A total of 20 strains from the DuCV-positive flock were sequenced. The total sequence length was 1987-1996 nt, and the sequences shared 82% (JX499186, DuCV2 from Sichuan province, China) to 99.7% (KY328304, DuCV1 from Shandong Province, China) sequence identity with DuCV sequences available in GenBank. Hyper-variable regions were mainly located in open reading frame (ORF)2, ORF3 and intergenic regions. The tertiary structure of ORF2 from four provinces (Henan, Anhui, Zhejiang and Fujian) in China showed a canonical viral jelly roll and the antigenic epitope of ORF2 located in the bulge of the protein surface. Overall, 15 of the 20 DuCV strains are possibly derived through inter-genotypic and intragenotypic recombination. Based on sequence and phylogenetic analyses, six strains from Fujian Province clustered into a novel genotype-DuCV-1d. These findings may enrich our understanding of DuCV evolution and circulation and lay the foundation for vaccine strain selection.

Characterization and genomic analysis of emerging astroviruses causing fatal gout in goslings.

Since February 2017, severe outbreaks of fatal gout caused by novel gosling astroviruses (GoAstVs) have occurred in several Chinese provinces, causing a considerable economic impact on the poultry industry. To assess the infection status of GoAstVs causing gout, 165 clinical samples were collected from goslings from seven farms located in different Chinese provinces, and they were screened for viral infection. Seven GoAstV strains were completely sequenced. The positive infection rates of GoAstV, goose parvovirus, reovirus, goose haemorrhagic polyomavirus and Tembusu virus were 100%, 9.69%, 3.64%, 0% and 0%, respectively, indicating the role of GoAstV in gout. The genomes of all seven GoAstV strains were 7170-nt long and encoded three open reading frames (ORFs), namely, ORF1a, ORF1b and ORF2. Sequence and phylogenetic analyses of the seven GoAstV strains showed that these were avastroviruses and were closely related to viruses classified within Avastrovirus 3 and turkey astrovirus 2. Moreover, the mutation rates of ORF1a and ORF2 were high, and ORF1a was highly mutated at amino acid loci 545-580. The tertiary structure of the mutated ORF2 protein was smooth, and its antigenic epitope was highly mutated, which may be related to the pathogenicity of the virus and caused by antibody pressure from the host. These findings enrich our understanding of the evolution of novel GoAstVs causing gout and their circulation as well as lay the foundation for the selection of vaccine strains.