Yaks provide necessities such as meat and milk for Tibetans living at high altitudes on and around the Qinghai-Tibetan Plateau. Enterococci are ubiquitous members of the animal gut microbiota that can cause biofilm-associated opportunistic infections. Meanwhile, multidrug-resistant Enterococcus also poses a serious threat to public health. This study aims to characterize antibiotic resistance, virulence genes, and biofilm formation of enterococci from yaks. From April 2018 to July 2019, we collected 395 fecal samples of yaks in Aba Tibetan Autonomous Prefecture, China. Enterococci isolated from the samples were identified and classified according to the 16S rDNA sequence. The antibiotic resistance of each isolate was detected according to the Kirby-Bauer disk diffusion method, and antibiotic resistance genes were detected by polymerase chain reaction (PCR) and sequencing. Enterococcal biofilms were assessed using standard procedures. Different virulence genes were detected by PCR and sequencing. In total, 381 enterococci strains were recovered, with Enterococcus faecalis (41.99%) and Enterococcus faecium (37.80%) being the predominant species. Many isolates were multidrug- resistant (60.37%) and showed a high resistance rate to rifampicin (64.30%) and tetracycline (61.54%). We also detected various antimicrobial resistance (AMR) genes in the tested strains. The E. faecalis strains had higher frequency of biofilm formation and virulence genes than other enterococcal species. This is the first report that shows yaks are repositories for drug-resistant enterococci with virulent determinants and biofilms that may spread into humans and to environment. This study also provides useful data suggesting that enterococci may pose a potential health risk to yaks. Therefore, active surveillance of AMR and pathogenesis in enterococci from yaks is urgently warranted.

Avian infectious bronchitis has caused huge economic losses in the poultry industry. Previous studies have reported that infectious bronchitis virus (IBV) infection can produce cytopathic effects (CPE) and apoptosis in some mammalian cells and primary cells. However, there is little research on IBV-induced immune cell apoptosis. In this study, chicken macrophage HD11 cells were established as a cellular model that is permissive to IBV infection. Then, IBV-induced apoptosis was observed through a cell viability assay, morphological changes, and flow cytometry. The activity of caspases, the inhibitory efficacy of caspase-inhibitors and the expression of apoptotic genes further suggested the activation of apoptosis through both intrinsic and extrinsic pathways in IBV-infected HD11 cells. Additionally, ammonium chloride (NH₄Cl) pretreated HD11 cells blocked IBV from entering cells and inhibited IBV-induced apoptosis. UV-inactivated IBV also lost the ability of apoptosis induction. IBV replication was increased by blocking caspase activation. This study presents a chicken macrophage cell line that will enable further analysis of IBV infection and offers novel insights into the mechanisms of IBV-induced apoptosis in immune cells.

Recombination among porcine reproductive and respiratory syndrome viruses (PRRSVs) is thought to contribute to the emergence of new PRRSV variants. In this study, two newly emerged PRRSV strains, designated SCcd16 and SCya17, are isolated from lung tissues of piglets in Southwestern China. Genome comparative analysis reveals that SCcd16/SCya17 exhibit 93.1%/93.2%, 86.9%/87.0%, 85.3%/85.7%, and 83.6%/82.0% nucleotide similarity to PRRSVs JXA1, VR-2332, QYYZ and NADC30, respectively. They only exhibit 44.8%/45.1% sequence identity with LV (PRRSV-1), indicating that both emergent strains belong to the PRRSV-2 genotype. Genomic sequence alignment shows that SCcd16 and SCya17 have the same discontinuous 30-amino acid (aa) deletion in Nsp2 of the highly pathogenic Chinese PRRSV strain JXA1, when compared to strain VR-2332. Notably, SCya17 shows a unique 5-nt deletion in its 3’-UTR. Phylogenetic analysis shows that both of the isolates are classified in the QYYZ-like lineage based on ORF5 genotyping, whereas they appear to constitute an inter-lineage between JXA1-like and QYYZ-like lineages based on their genomic sequences. Furthermore, recombination analyses reveal that the two newly emerged PRRSV isolates share the same novel recombination pattern. They have both likely originated from multiple recombination events between lineage 8 (JXA1-like), lineage 1 (NADC30-like), and lineage 3 (QYYZ-like) strains that have circulated in China recently. The genomic data from SCcd16 and SCya17 indicate that there is on going evolution of PRRSV field strains through genetic recombination, leading to outbreaks in the pig populations in Southwestern China.

Avian infectious bronchitis (IB), caused by avian infectious bronchitis virus (IBV), is an acute and highly contagious disease that is extremely harmful to the poultry industry throughout the world. The cross-using of different attenuated live vaccine strains has led to the occurrence of diverse IBV serotypes. In this study, we isolated an IBV strain from a chicken farm in southwest China and designated it CK/CH/SCMY/160315. Construction of a phylogenetic tree based on full S1 gene sequence analysis suggested that CK/CH/SCMY/160315 bears similarity to GI-28, and further comparison of S1 amino acid residues revealed that CK/CH/SCMY/160315 showed mutations and deletions in many key positions between LDT3-A and other GI-28 reference strains. Importantly, CK/CH/SCMY/160315 was identified as a novel recombinant virus derived from live attenuated vaccine strains H120 (GI-1), 4/91 (GI-13), LDT3-A (GI-28), and the field strain LJL/08-1 (GI-19), identifying at least 5 recombination sites in both structural and accessory genes. Pathogenicity analysis indicated that CK/CH/SCMY/160315 caused listlessness, sneezing, huddling, head shaking, and increased antibody levels in the inoculated chickens. To further describe pathogenicity of this novel strain, we assessed viral load in different tissues and conducted hematoxylin and eosin (HE) staining of the trachea, lungs and kidneys. Our results provide evidence for the continuing evolution of IBV field strains via genetic recombination and mutation, leading to outbreaks in the vaccinated chicken populations in China.

Avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is the causative agent of infectious bronchitis (IB) that has brought great threat and economic losses to the global poultry industry. Rapid and accurate diagnostic methods are very necessary for effective disease monitoring. At the present study, we screened a novel nanobody against IBV-N protein for development of a rapid, simple, sensitive, and specific competitive ELISA for IBV antibody detection in order to enable the assessment of inoculation effect and early warning of disease infection. Using the phage display technology and bio-panning, we obtained 7 specific nanobodies fused with horseradish peroxidase (HRP) which were expressed in culture supernatant of HEK293T cells. Out of which, the nanobody of IBV-N-Nb66-vHRP has highly binding with IBV-N protein and was easily blocked by the IBV positive serums, which was finally employed as an immunoprobe for development of the competitive ELISA (cELISA). In the newly developed cELISA, we reduce the use of enzyme-conjugated secondary antibody, and the time of whole operation process is approximately 1 h. Moreover, the IBV positive serums diluted at 1:1000 can still be detected by the developed cELISA, and it has no cross reactivity with others chicken disease serums including Newcastle disease virus, Fowl adenovirus, Avian Influenza Virus, Infectious bursal disease virus and Hepatitis E virus. The cut-off value of the established cELISA was 36%, and the coefficient of variation of intra- and inter-assay were 0.55–1.65% and 2.58–6.03%, respectively. Compared with the commercial ELISA (IDEXX kit), the agreement rate of two methods was defined as 98% and the kappa value was 0.96, indicating the developed cELISA has high consistency with the commercial ELISA. Taken together, the novel cELISA for IBV antibody detection is a simple, rapid, sensitive, and specific immunoassay, which has the potential to rapidly test IBV antibody contributing to the surveillance and control of the disease.