Changes of intestinal microbiota in the giant salamander (Andrias davidianus) during growth based on high-throughput sequencing.

Despite an increasing appreciation of the importance of host-microbe interaction in healthy growth, information on gut microbiota changes of the Chinese giant salamander (Andrias davidianus) during growth is still lacking. Moreover, it is interesting to identify gut microbial structure for further monitoring A. davidianus health. This study explored the composition and functional characteristics of gut bacteria in different growth periods, including tadpole stage (ADT), gills internalization stage (ADG), 1 year age (ADY), 2 year age (ADE), and 3 year age (ADS), using high-throughput sequencing. The results showed that significant differences were observed in microbial community composition and abundance among different growth groups. The diversity and abundance of intestinal flora gradually reduced from larvae to adult stages. Overall, the gut microbial communities were mainly composed of Fusobacteriota, Firmicutes, Bacteroidota, and Proteobacteria. More specifically, the Cetobacterium genus was the most dominant, followed by Lactobacillus and Candidatus Amphibiichlamydia. Interestingly, Candidatus Amphibiichlamydia, a special species related to amphibian diseases, could be a promising indicator for healthy monitoring during A. davidianus growth. These results could be an important reference for future research on the relationship between the host and microbiota and also provide basic data for the artificial feeding of A. davidianus.

The central role of GmGLP20.4 in root architecture modifications of soybean under low-nitrogen stress.

KEY MESSAGE: The GmGLP20.4 candidate gene plays an important role to improve soybean root architecture under low-nitrogen stress. The results lay the foundation for breeding low-nitrogen-tolerant soybean. Roots are fundamentally important for plant growth and development, facilitating water and nutrient uptake. Various abiotic and biotic factors significantly affect the root system architecture, especially low nitrogen (LN), but the molecular mechanism remains unclear. In this study, we identified GmGLP20.4, a germin-like protein (ubiquitous plant glycoproteins belonging to the Cupin superfamily) crucial for lateral root development and highly induced by LN stress in lateral roots of soybean. GmGLP20.4 overexpression increased root biomass through development of an improved root system in soybean under LN, whereas a significant decrease in root biomass was observed in the gmglp20.4 knockout mutant. Overexpression of GmGLP20.4 improved plant growth and root architecture in transgenic tobacco (Nicotiana tabacum) under LN. Natural variation of the GT-1 cis-element in the promoter (T to A) of GmGLP20.4 was strongly associated with its expression level under LN, and significantly increased LN-sensitive variation (type A) was observed in wild soybean compared to that in elite cultivars. Thus, type A variation in the promoter of GmGLP20.4 may have been a site of artificial selection during domestication. The GmGT1-16g gene was highly expressed under LN and showed an expression pattern opposite to that of GmGLP20.4. A luciferase complementation imaging assay revealed that the GmGLP20.4 promoter specifically binds to GmGT1-16g. In conclusion, GmGLP20.4 is involved in soybean root development and the natural variation of its promoter will be useful in modern intercropping systems or to improve nitrogen-use efficiency.

The Regulation of Staphylococcus aureus-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells.

Mastitis, an inflammatory disease, causes severe economic loss in the dairy industry, which is mainly infected by bacteria. Staphylococcus aureus (S. aureus), the major pathogenic microorganism, derived from lipoteichoic acid (LTA) has been identified to activate inflammatory responses, but the cellular or intercellular regulatory mechanism is unclear. This study mainly focused on the effects of LTA in bovine mammary epithelial cells (Mac-T) and elaborated the regulation of microRNAs (miRNAs). The results showed that LTA enhanced the messenger RNA (mRNA) expression and production of tumor necrosis factor α (TNF-α) and interleukin (IL)-6. Furthermore, LTA could activate Toll-like receptor (TLR)2/MyD88-mediated phosphoinositide 3-kinase (PI3K)/AKT pathway, and TLR2 plays a pivotal role in LTA-induced inflammatory responses. The results of qRT-PCR showed that miRNA levels increased and reached the highest at 3 h and then gradually decreased over time in Mac-T cells. In exosomes, the levels of 11 and three miRNAs were upregulated and downregulated at 24 h, respectively. In addition, miR-23a showed the highest increase in Mac-T cells treated with LTA and targeted PI3K to regulate inflammatory responses. Furthermore, Mac-T cell-derived exosomes were identified to play a cell-cell communication by promoting M1 polarization of bovine macrophages. In summary, our study demonstrated that LTA could activate inflammatory responses via TLR2/MyD88/PI3K/AKT signaling pathway, and miR-23a inhibited it by targeting PI3K. Furthermore, we found that Mac-T cell-derived exosomes might be associated with inflammatory responses by promoting M1 polarization of bovine macrophages.

Comparative transcriptome analyses reveal the genetic basis underlying the immune function of three amphibians' skin.

Skin as the first barrier against external invasions plays an essential role for the survival of amphibians on land. Understanding the genetic basis of skin function is significant in revealing the mechanisms underlying immunity of amphibians. In this study, we de novo sequenced and comparatively analyzed skin transcriptomes from three different amphibian species, Andrias davidianus, Bufo gargarizans, and Rana nigromaculata Hallowell. Functional classification of unigenes in each amphibian showed high accordance, with the most represented GO terms and KEGG pathways related to basic biological processes, such as binding and metabolism and immune system. As for the unigenes, GO and KEGG distributions of conserved orthologs in each species were similar, with the predominantly enriched pathways including RNA polymerase, nucleotide metabolism, and defense. The positively selected orthologs in each amphibian were also similar, which were primarily involved in stimulus response, cell metabolic, membrane, and catalytic activity. Furthermore, a total of 50 antimicrobial peptides from 26 different categories were identified in the three amphibians, and one of these showed high efficiency in inhibiting the growth of different bacteria. Our understanding of innate immune function of amphibian skin has increased basis on the immune-related unigenes, pathways, and antimicrobial peptides in amphibians.

Development of an ELISA based on a multi-fragment antigen of infectious bronchitis virus for antibodies detection.

OBJECTIVES: To develop a cost-effective ELISA for detection of antibodies against infectious bronchitis virus (IBV) by using a multi-fragment protein as coating antigen. RESULTS: A multi-fragment antigen, termed BE, which was composed of eight antigenic fragments selected from the three major proteins (S, M, and N) of IBV, was expressed in Escherichia coli. The entire protein had a molecular weight of 61.5 kDa. In addition to it, a smaller truncated protein was also produced; both could react with IBV-positive serum. Next, an indirect ELISA (BE-ELISA) was developed. Coefficients of variation of this assay were lower than 10 %, and no cross-reactivity between the coated antigen BE and antiserum against newcastle disease virus, avian influenza virus, or infectious bursal disease virus was observed. The performance of BE-ELISA was evaluated, and showed 95.4 % coincidence ratio with the whole virus based-ELISA (IDEXX). CONCLUSIONS: The multi-fragment antigen (BE) may represent a promising alternative to the whole virus without safety problems, and this newly established ELISA provides an effective method for anti-IBV antibody detection.

Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus.

An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166-247 aa, S1 gene; 501-515 aa, S1 gene; 8-30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 µg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD450 value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.

Comparative dynamic distribution of avian infectious bronchitis virus M41, H120, and SAIBK strains by quantitative real-time RT-PCR in SPF chickens.

Avian infectious bronchitis is an acute, highly contagious disease of chickens. To study the differences of dynamic distribution between nephropathogenic infectious bronchitis virus (IBV) strains such as SAIBK and other strains (the M41 and H120 strains), relative quantitative real-time reverse transcription-polymerase chain reaction was developed by housekeeping gene selection. Glyceraldehyde-3-phosphate dehydrogenase and Ubiquitin were chosen for normalization in this experimental set. Then nine tissues, the trachea, thymus, liver, spleen, lungs, kidney, pancreas, proventriculus, and bursa of Fabricius, were analyzed and compared to determine the tropism of IBV infection. In this research, the kidney and the lung were established of the most sensitive organs in IBV infection. The pancreas and the liver are candidates for antigen detection. The trachea and the spleen can be used as references for histological diagnosis, but they are not suitable for antigen detection; proventriculus might be an important target in IBV infection; the thymus and the bursa of Fabricius were not sensitive organs in IBV infection.

Detection of clinically important ß-lactamases in commensal Escherichia coli of human and swine origin in western China.

Data correlating ß-lactamases found in commensal Escherichia coli of human and animal origin are limited. In this study, 447 commensal E. coli isolates from the faeces of humans and swine (280 human isolates from four hospitals and 167 swine isolates from seven farms) were collected between September 2006 and January 2009 in western China. For extended-spectrum ß-lactamase (ESBL)-producing and other cephalosporin-resistant isolates, the relevant ß-lactamase genes (bla(TEM), bla(SHV), bla(CTX-M-1/2/9) group, bla(CMY-2) and bla(KPC)) were detected by PCR analysis. Of the 447 isolates tested, 120 (26.8 %) were confirmed as producing ESBL. Among these, 70 and 40 human isolates carried a member of the bla(CTX-M-1 )group (13 bla(CTX-M-3), 21 bla(CTX-M-15), four bla(CTX-M-22), eight bla(CTX-M-28), four bla(CTX-M-36), 15 bla(CTX-M-55) and five bla(CTX-M-69)) or bla(SHV) (14 bla(SHV-2), seven bla(SHV-5), ten bla(SHV-12), five bla(SHV-57) and four bla(SHV-97)),respectively, whilst six and four swine isolates carried a member of the bla(CTX-M-1 )group (one bla(CTX-M-15) and five bla(CTX-M-22)) or bla(SHV) (three bla(SHV-2) and one bla(SHV-12)), respectively. Furthermore, 59 human and swine isolates and seven human isolates carried bla(CMY-2) and bla(KPC), respectively. These findings indicate that the bla(CTX-M-1) group, including the novel variant bla(CTX-M-69), and bla(SHV) are the predominant ESBL genes in both humans and swine in western China, and bla(CMY-2) is also common in both groups. The carriage rates of broad-spectrum ß-lactamases among commensal E. coli was much lower in swine than in humans, suggesting that ß-lactamase genes have not established themselves in animal ecosystems in western China.

Complete genome sequence and recombination analysis of infectious bronchitis virus attenuated vaccine strain H120.

The strain H120 of infectious bronchitis virus (IBV) is one of the earliest and representative attenuated live Infectious Bronchitis vaccine strains. To investigate the genomic feature of H120 and further understand its role in the epidemiology of IBV, complete genome of H120 was sequenced and compared with sequences of other IBV strains by phylogenetic and recombination analysis. The complete genome of H120 is 27631 nucleotides in length and has a similar structure with that of Beaudette strain. We found that strain ZJ971 is probably a virulence revertant of H120. Nine amino acids changes and a three-nucleotide deletion were identified in ZJ971. Besides, potential recombination events associated with H120 were found in five IBV strains including H52, KQ6, SAIBK, Ark DPI 11, and Ark DPI 101. This study suggested that H120 might have contributed to the emergence of new IBV variants through both virulence reversion and recombination.

An effective method for isolation of DNA from pig faeces and comparison of five different methods.

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and beta-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp DNA Stool Mini Kit.