To establish a multiplex PCR for simultaneous detection of Escherichia coli (E. coli), Salmonella, Klebsiella pneumoniae (K. pneumoniae), and Staphylococcus aureus (S. aureus), four pairs of specific primers were designed according to the conservative regions of phoA gene for E. coli, invA gene for Salmonella, khe gene for K. pneumoniae, nuc gene for S. aureus. The quadruple PCR system was established through optimization of multiplex PCR and detection of specificity, sensitivity, and stability. The results showed that target gene bands of E. coli (622 bp), Salmonella (801 bp), K. pneumoniae (303 bp), and S. aureus (464 bp) could be amplified by this method specifically and simultaneously from the same sample containing the four pathogens, with a detection sensitivity of 100 pg/μL. Meanwhile, no bands of common clinical bacteria, including Clostridium perfringens, Pseudomonas aeruginosa, Pasteurella multocida, Streptococcus pneumoniae, Streptococcus pneumoniae, Proteus mirabilis, Staphylococcus sciuri, Staphylococcus pseudintermedius, Acinetobacter baumannii, Enterococcus faecalis, and Bacillus subtilis were amplified. In addition, 380 tissue samples were detected by multiplex and single PCR established in current study, respectively. Among the 368 carcass samples, positive detection rates of E. coli, K. pneumoniae, Salmonella, and S. aureus were 33.7, 12.0, 10.6, and 13.9%. Among the 12 visceral tissue samples, positive detection rates of E. coli, K. pneumoniae, Salmonella, and S. aureus were 41.7, 25.0, 16.7, and 8.3%, respectively. Positive detection rates of multiplex PCR were consistent with that of single PCR. Compared with single PCR, the multiplex PCR method had the advantages of time-saving, high specificity and high sensitivity. The results showed that the minks in these farms had mixed infection of these four pathogens, and the method established in this study could be applied to the rapid and accurate detection and identification of these four bacteria. In conclusion, the multiplex PCR method has stable detection results, good repeatability, and short detection time. It is suitable for the rapid and accurate detection of four kinds of bacteria above the carcass of fur animals, which could be suitable in microbial epidemiology investigation. It can provide a reliable technical reference for rapid clinical diagnosis and detection.

Background: Cha-hua (Camellia reticulata) is one of China’s traditional ornamental flowers developed by the local people of Yunnan Province. Today, more than 500 cultivars and hybrids are recognized. Many ancient camellia trees still survive and are managed by local peopl. A few records on cha-hua culture exist, but no studies expound the interaction between C. reticulata and traditional culture of ethnic groups. The contribution of traditional culture of different nationalities and regions to the diversity of Camellia reticulate is discussed. Methods: Ethnobotanical surveys were conducted throughout Central and Western Yunnan to investigate and document the traditional culture related to Camellia reticulata. Five sites were selected to carry out the field investigation. Information was collected using participatory observation, semi-structured interviews, key informant interviews, focus group discussions, and participatory rural appraisal (PRA). Results: Most of the ancient camellia trees were preserved or saved in the courtyards of old buildings and cultural or religious sites. Religion-associated culture plays an important role in C. reticulata protection. In every site we investigated, we found extensive traditional culture on C. reticulata and its management. These traditional cultures have not only protected the germplasm resources of C. reticulata, but also improved the diversity of Camellia cultivars. Conclusions: There are abundant and diverse genetic resources of cha-hua, Camellia reticulata in Yunnan. Cha-hua is not only an ornamental flower but also has been endowed with rich spiritual connotation. The influence of traditional culture had improved the introduction and domestication of wild plants, breeding and selection of different varieties, and the propagation and dissemination of the tree in Yunnan. However, either some ancient cha-hua trees or their associated traditional culture are facing various threats. The old cha-hua trees and the ethnic camellia culture should be respected and protected since they have made great contributions in the history, and will make more contributions in the future.

This study focuses on the immunoregulatory effects of chicken TRIM25 on the replication of subgroup A of avian leukosis virus (ALV-A) and the MDA5-mediated type I interferon response. The ALV-A-SDAU09C1 strain was inoculated into DF1 cells and 1-day-old SPF chickens, and the expression of TRIM25 was detected at different time points after inoculation. A recombinant overexpression plasmid containing the chicken TRIM25 gene (TRIM25-GFP) was constructed and transfected into DF1 cells to analyse the effects of the overexpression of chicken TRIM25 on the replication of ALV-A and the expression of MDA5, MAVS and IFN-β. A small interfering RNA targeting chicken TRIM25 (TRIM25-siRNA) was prepared and transfected into DF1 cells to assess the effects of the knockdown of chicken TRIM25 on the replication of ALV-A and the expression of MDA5, MAVS and IFN-β. The results showed that chicken TRIM25 was significantly upregulated at all time points both in ALV-A-infected cells and in ALV-A-infected chickens. Overexpression of chicken TRIM25 in DF1 cells dramatically decreased the antigenic titres of ALV-A in the cell supernatant and upregulated the relative expression of MDA5, MAVS and IFN-β induced by ALV-A or by poly(I:C); in contrast, knockdown of chicken TRIM25 significantly increased the antigenic titres of ALV-A and downregulated the relative expression of MDA5, MAVS and IFN-β. It can be concluded that chicken TRIM25 can inhibit the replication of ALV-A and upregulate the MDA5 receptor-mediated type I interferon response in chickens. This study can help improve the understanding of the antiviral activities of chicken TRIM25 and enrich the knowledge of antiviral responses in chickens.

Subgroup A of the avian leukosis virus (ALV-A) can cause severe pathological lesions and death in infected chickens, and its reported hosts have increased recently. To assess the susceptibility of adult chickens, quails, and pigeons to ALV-A, three sets of 250-day-old birds were intraperitoneally inoculated with ALV-A. Viremia and cloacal virus shedding were dynamically detected using an immunofluorescence assay (IFA), ALV-P27 antigen ELISA or RT-PCR; pathological lesions were assessed using tissue sections; ALV-A in tissues was detected by IFA; and ALV-A antibody responses were detected using antibody ELISA kits and an immune diffusion test. The results indicated that persistent viremia occurred in 80% (8/10) of infected chickens, and transient viremia occurred in 17% (2/12) of infected quails, but no viremia occurred in infected pigeons. Cloacal virus shedding occurred intermittently in 80% (8/10) of infected chickens and in 8% (1/12) of infected quails but did not occur in infected pigeons. Severe inflammatory pathological lesions occurred in the visceral tissues of most infected chickens, and mild lesions occurred in a few of the infected quails, but no pathological lesions occurred in the infected pigeons. The ALV-A virus was detected in the visceral tissues of most infected chickens but not in the infected quails and pigeons. Obviously different ALV-A antibody responses occurred in the infected chickens, quails and pigeons. It can be concluded that adult chickens, quails and pigeons have dramatically different susceptibilities to ALV-A. This is the first report on artificial infection by ALV-A in different birds.