In order to explore the pathogen of emulsification disease in Chinese mitten crab (Eriocheir sinensis) in Tianjin district, the epidemiological investigation, pathogen isolation, 18S rDNA gene sequence analysis, artificial infection test, and immunohistochemistry (IHC) analysis were carried out for identification of the pathogen, tissue distribution and temporal variation characteristics in this study. The results showed that the main clinical symptoms of naturally infected crabs included slow or almost no response to external stimuli, emulsify hemolymph accumulation in the cephalothorax cavity, opaque and whitish muscles at cephalothorax and joint membrane at the base of claw joints, and white semi-fluid emulsification of the hepatopancreas. The fungal strain P13, isolated from the diseased crab, was identified as Metschnikowia bicuspidata according to the phylogenetic analysis based on 18S rDNA gene sequences. Artificial infection test results showed that P13 could infect healthy Eriocheir sinensis under the laboratory condition, with cumulative mortality of (55.0±2.4)%, causing clinical symptoms similar to those found in the naturally infected crabs, and the Metschnikowia bicuspidata can be isolated from the experimental crabs again. IHC analysis results indicated that P13 infected various tissues of Eriocheir sinensis, including hepatopancreas, hindgut, gill, heart, and muscle, causing pathological damages in different degrees and temporal variation characteristics in the targeted tissues. In conclusion, Metschnikowia bicuspidata was the pathogen of the emulsification disease in Eriocheir sinensis. This study preliminarily revealed the main target tissues of Metschnikowia bicuspidata and the characteristics of tissue distribution and temporal variation, which provides a theoretical basis for clarifying the pathogenesis of Metschnikowia bicuspidata, and offers technical support for the control and prevention of emulsification disease in Eriocheir sinensis.
Krait software was used to analyze the distribution characteristics of perfect microsatellites in the whole genome of Protosalanx chinensis, which was published in 2020 with a higher degree of splicing, and to develop polymorphic microsatellite DNA (also known as simple sequence repeat) markers. The results showed that a total of 587 554 perfect microsatellite loci were obtained in the whole genome of P. chinensis, with a total sequence length of 11 803 017 bp, accounting for 2.53% of the whole genome length. Among six repeat types of microsatellites, the number of dinucleotide was the largest (401 585, accounting for 68.35%). In the 99 pairs of primers designed for microsatellite loci， 39 were polymorphic. Among them, 14 microsatellite markers with favorable polymorphism were selected to test one representative population selected from each of the migratory, landlocked, and introduced populations. The results indicated that 14 microsatellite markers with favorable polymorphism could achieve effective amplification in the three representative populations. The genetic diversity and genetic structure of the three populations were analyzed, and it was found that the migratory population (Chongming Island population) had abundant genetic variation (the mean expected heterozygosity is 0.614, and the mean polymorphism information content is 0.576), which could be clustered into a genetic group different from the freshwater populations [including Taihu Lake population (landlocked) and Lianhuan Lake population (introduced)], and there were large genetic distance and extremely high level of genetic differentiation level between them [the genetic differentiation index (Fst) is higher than 0.25, P＜0.05]. The genetic variations between the two freshwater populations (Taihu Lake and Lianhuan Lake populations) were relatively scarce and the genetic distance between them was small. Although there was significant genetic differentiation between them, the genetic differentiation level was relatively low (Fst=0.102, P＜0.05). These results indicate that the migratory population has potential conservation value of germplasm resource, which provide basis for the development of microsatellite markers and construction of genetic maps, and furthermore provide references for the subsequent evaluation of large-scale population germplasm resources of P. chinensis.
To prepare the specific antibodies against nucleocapsid (N) protein of feline coronavirus (FCoV), the full-length cDNA of N gene was amplified from type ⅠFCoV (ZJU1709) and subcloned into pCold TF prokaryotic expression vector. The recombinant N protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) at low temperature and affinity-purified using nickel column. The rabbit polyclonal antiserum was prepared using the purified recombinant N protein as antigen. Finally, the enzyme-linked immunosorbent assay (ELISA), Western blotting (WB) and indirect immunofluorescence assay (IFA) were applied to identify the reactivity of rabbit polyclonal antiserum. The results showed that the soluble recombinant N protein with a molecular weight of about 100 kDa was successfully induced and further purified under non-denaturing condition at a concentration of 1.4 mg/mL. The ELISA titer of the prepared rabbit polyclonal antiserum of N protein can reach 1×106, and it can react with the recombinant Flag-N eukaryotic protein and N protein in FCoV-infected cells by WB and IFA. Cross-reactivity analysis showed that the polyclonal antibody can react with N proteins of alphacoronavirus, including various subtypes of FCoV, as well as canine coronavirus (CCoV), porcine transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV), but failed to react with N proteins of betacoronavirus of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and gammacoronavirus of infectious bronchitis virus (IBV). In summary, the soluble recombinant N protein expressed by using pCold TF prokaryotic system showed strong immunogenicity. The rabbit polyclonal antiserum against N protein of FCoV can recognize N proteins of multiple species from alphacoronavirus, but showed no cross-reactivity with N proteins of betacoronavirus and gammacoronavirus. This antibody facilitates to study the replication mechanism of FCoV and develop efficient methods for antigen or antibody detection.
The aim of this study was to systematically investigate the differences in the soluble expression level of the African swine fever virus (ASFV) CD2v protein by different prokaryotic expression vectors, and the immunoreactivities of the inclusion body and soluble CD2v proteins were compared using clinical anti-ASFV antibody-positive sera. Five prokaryotic expression vectors, namely, pCold-TF, pET28a, pMAL-C6T, pGEX-4T-1 and pET32a, were utilized to express the CD2v protein without the signal peptide and transmembrane region, respectively. The inclusion body CD2v protein expressed by the pET28a vector and the soluble CD2v protein expressed by the pCold-TF vector were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, and the immunoreactivity of the purified proteins was detected by indirect enzyme-linked immunosorbent assay (ELISA). The results showed that the CD2v protein expressed by the pCold-TF vector was soluble mainly, while the CD2v protein expressed by the pMAL-C6T vector was insoluble (inclusion body) and soluble, and the CD2v protein expressed by the other vectors was mainly as inclusion body. The indirect ELISA results for clinical anti-ASFV antibody-positive sera showed that the immunoreactivity of soluble protein was significantly better than that of the inclusion body protein (P＜0.05). The trigger factor (TF) tag of pCold-TF promoted the soluble expression of the CD2v protein, and the immunoreactivity of the expressed protein was greater than that of the inclusion body protein. This study lays the foundation for further immunogenicity research on the CD2v protein and provides a candidate strategy for the soluble expression of other important antigens.
This study aims to use microbial fermentation technology to improve the utilization of soybean residues and to develop a new type of fermented feed with superior quality and competitive price. In this experiment, fermented soybean residues were used to feed 1 day-of-age Xianju chickens, lasting for 42 d. Five groups were set up, including the control group T1, fed with the basal diets; the antibiotic group T2, fed with the basal diets supplemented with 40 mg/kg methylene salicylic acid bacitracin; the treatment groups T3, T4, and T5, fed with the basal diets in which 2%, 4%, and 6% soybean meal were replaced by fermented soybean residues, respectively. The results were shown as follows. 1) Compared with the control group T1, the 42 day-of-age body mass and average daily gain of Xianju chickens were significantly higher in each group, and the feed to gain ratio was significantly lower in T4 group (P＜0.05). 2) Compared with the control group, the albumin content was extremely significantly higher in T4 group (P＜0.01), and the superoxide dismutase activity was extremely significantly higher in T2 and T5 groups (P＜0.01), while the malondialdehyde content was extremely significantly lower in T2 group (P＜0.01). 3) Compared with the control group, the apparent digestibility of crude protein and crude fiber was significantly increased in T4 group (P＜0.05) and the apparent digestibility of crude protein was extremely significantly increased in T5 group (P＜0.01); the duodenal amylase activity in T4 group (P＜0.05), the duodenal protease activities in T4 and T5 groups (P＜0.01), and the chymotrypsin activities in T2, T3, and T5 groups (P＜0.05) were significantly improved. In conclusion, replacing 4% soybean meal in the basal diets with an equal amount of fermented soybean residues has the best feeding effect, which could significantly promote the growth performance, apparent digestibility of crude protein and crude fiber, and digestive enzyme activities in vivo of Xianju chickens, improve their serum indexes, and has the potential to replace antibiotics.
In order to understand the changes and characteristics of microbial communities during early development of Protosalanx chinensis, samples were collected at five developmental periods, which are the embryonic period (heartbeat stage, XT), the endogenous nutrition period (the first day after hatching, H1), the mixed nutrition period (the fourth day after hatching, H4), the open feeding period (the seventh day after hatching, H7), and the exogenous nutrition period (the tenth day after hatching, H10). Microbial community succession during early development was observed by 16S rRNA gene sequencing technology, especially the characteristics and key microbial genera before and after feeding. Combined with the transcriptome data of the same batch of samples, the microbial genera related to immunity and metabolism were analyzed based on the association network method. The results showed that there were significant differences in β diversity among the different periods at the early developmental stage (P＜0.001). The dominant bacteria in the XT period were Flavobacterium and Chryseobacterium, and the dominant bacterium in the H1 period was Pseudomonas. The main bacteria in the H4 period were Flavobacterium and Pseudomonas. The main bacteria in H7 and H10 periods were Flectobacillus and Pseudomonas. The abundance of Pseudomonas was stable at all developmental periods. Node bacteria such as Flectobacillus were significantly associated with the expression of various immune and metabolic genes. This study has obtained the microbial succession information of the early developmental stage of P. chinensis for the first time, and screened out the dominant bacteria and node bacteria, which will provide references and ideas for the scaled cultivation of P. chinensis fry.
The purpose of this study was to screen key mRNAs and long non-coding RNAs (lncRNAs) that affect gonadal development in duck embryos and to explain scientifically gonadal development defects in the mulard duck. Three male embryonic gonadal tissues of the mulard duck (BF1, BF2, BF3) and the muscovy duck (F1, F2, F3) were collected to extract RNA and perform high-throughput sequencing, and differential genes and lncRNAs were screened to predict target genes and perform functional annotations. Finally, the sequencing data were verified by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The results showed that a total of 1 109 differentially expressed genes were screened from the gonadal tissues of the mulard duck and the muscovy duck. Compared with the muscovy duck, 857 genes were up-regulated and 252 genes were down-regulated in the mulard duck. Among them, the aldo-keto reductase family 1 member D1 gene (AKR1D1), 17β-hydroxysteroid dehydrogenase 3 gene (17β-HSD3), and cholesterol side-chain cleavage enzyme gene (P450scc) may be related to gonadal differentiation and development in the mulard duck. Meanwhile, 733 significantly differentially expressed lncRNAs were obtained. Compared with the muscovy duck, a total of 660 lncRNAs were significantly up-regulated and 73 lncRNAs were significantly down-regulated in the mulard duck. Target gene prediction analysis showed that a total of 136 down-regulated lncRNAs and 893 up-regulated lncRNAs may be involved in differential gene expression and had potential regulatory relationships, among which, TCONS_00246198 targeted 17β-HSD3, and TCONS_00229529 targeted tetraspanin-2 gene (TSPAN2), suggesting that the above lncRNAs may participate in duck embryonic gonadal development by targeting key genes. The qRT-PCR results showed that the expression levels of differential genes and lncRNAs were consistent with the expression trends in transcriptome sequencing, indicating that the data obtained by high-throughput sequencing are relatively reliable. RNA binding protein immunoprecipitation (RIP) assay results revealed that compared with IgG, the enrichment level of TCONS_00246198 reached 71.51 times. The above results indicate that TCONS_00246198 interacts directly or indirectly with the 17β-HSD3 protein, which means that they may have a targeting relationship. In summary, this study obtains a batch of key mRNAs and lncRNAs that may affect duck embryonic gonadal development, and it is speculated that the differential lncRNAs can regulate the expression of differential genes. This study provides a scientific basis for understanding the differences in duck embryonic gonadal development and the mechanisms of avian gonadal development.
This study aimed to investigate the changes in the composition and diversity of the rumen bacterial community in mid-lactation Holstein cows. In trial 1, a total of 20 healthy high-yielding Holstein cows at mid-lactation were continuously fed with the same basal diet for eight weeks and the rumen contents were collected on the 7th day at weeks zero, four, and seven. In trial 2, a total of 30 healthy high-yielding Holstein cows at mid-lactation were supplemented with 20 g/d rumen-protected methionine to the basal diet, and the rumen contents were collected on the 7th day at weeks zero and eight. The rumen contents collected at different time points were analyzed for changes in composition and diversity of the bacterial community, as well as differences in functional stability in both trials. The results showed that there were no significant differences in the alpha diversity, beta diversity, and functional stability of rumen bacteria at different time points (P＞0.05) in both trials. In trial 1, six and one differential bacteria, such as Actinobacteriota, Proteobacteria, Cyanobacteria, Chloroflexi, Synergistota, and Fibrobacterota, as well as Lachnospiraceae_NK3A20_group, were found in highly abundant bacteria at phylum and genus levels, respectively. In trial 2, three and one differential bacteria, such as Actinobacteriota, Spirochaetota, and Elusimicrobiota, as well as unclassified_f_Lachnospiraceae, were found in highly abundant bacteria at phylum and genus levels, respectively. It is indicated that the rumen bacterial community composition of Holstein cows at mid-lactation changed to a limited extent over time under the same feeding conditions, with no significant changes in their diversity and functional stability. Similar results were obtained when supplemented with rumen-protected methionine to the basal diet. In conclusion, the results suggested that the rumen microbial community composition and function of Holstein cows at mid-lactation were relatively stable, and there is no need to specifically consider the changes in rumen bacterial community structure and function along with lactation progress in the related experiments developed during the mid-lactation.
The non-structural protein 3 (Nsp3) of coronavirus, a component of the replication and transcription complex, is one of the potentially important antiviral targets. In this study, the transmembrane region, signal peptide, and epitope of Nsp3 were predicted, and then the region with better antigenicity (50-550 amino acids) of Nsp3 protein in a representative strain (WSU 79-1683) of type Ⅱ feline coronavirus (FCoV) was amplified by polymerase chain reaction. Subsequently, it was subcloned into pCOLD-TF prokaryotic expression vector. Under the low-temperature condition, the recombinant fusion protein His-Nsp3 with a molecular weight of about 130 kDa was successfully induced by isopropylthio-β-D-galactoside. The targeted recombinant protein His-Nsp3 was purified using a non-denaturing nickel affinity column, and the purified protein was used as an antigen to immunize BALB/c mice for preparing Nsp3 polyclonal antiserum. Western blotting (WB) and indirect immunofluorescence assay (IFA) results showed that Nsp3 polyclonal antiserum could specifically recognize Nsp3 protein in FCoV-infected cells. The subcellular localization of Nsp3 protein in FCoV-infected cells was studied by double-labeling IFA combined with laser confocal microscopy. The results showed that Nsp3 protein aggregated in FCoV-infected cells and co-localized with the endoplasmic reticulum. The specific antibody preparation and subcellular localization study of Nsp3 protein provided an important basis for further analysis of the biological function of Nsp3 protein.
The effects of aboveground parts of Tetrastigma hemsleyanum, T. hemsleyanum leaves (THL), on the growth performance, immune function and intestinal microflora of broilers were evaluated. A total of 240 broilers were randomly divided into four groups: the control group (fed with a basal diet) and low dose group, medium dose group and high dose group (fed with a basal diet supplemented with 1%, 3% and 5% THL powder, and denoted as THL-L, THL-M and THL-H, respectively). The body masses of broilers at 21 and 42 days of age in each group was measured, and the immune organ indexes and the contents of immunoglobulin A (IgA), IgG and IgM in serum and the relative abundance of intestinal microflora in cecum contents of broilers at 42 days of age were determined. The results showed that compared with the control group, the supplementation of THL powder significantly enhanced the average daily feed intake (P＜0.05) and average daily gain (P＜0.05) of broilers at 42 days of age, and the promoting effect of the THL-M group was the best. At the same time, compared with the control group, the thymus index and the bursa of fabricius index of broilers in THL-L and THL-M groups were significantly increased (P＜0.05). The contents of IgA and IgM in serum of broilers in THL-L, THL-M and THL-H groups were significantly higher than those in the control group (P＜0.05). However, IgG content in THL-M and THL-H groups was significantly higher than that in the control group (P＜0.05). The relative abundances of Lachnospiraceae and Clostridiaceae were significantly increased by the supplementation of appropriate amount of THL powder (3%) (P＜0.05). This study indicated that T. hemsleyanum leaf powder could significantly improve the immune function, the intestinal microflora composition and the growth performance of broilers, and the most significant effect was found with supplementation of 3% THL powder.
Apolipoprotein A-Ⅰ (Apo A-Ⅰ) plays important roles in the regulation of atherosclerosis, viral infections, lipid metabolism and other aspects. However, there are few studies on chicken Apo A-Ⅰ (chApo A-Ⅰ), and its biological function is not well understood. Based on the bioinformatics analysis of chApo A-Ⅰ, this study further performed the expression and purification of the recombinant protein of chApo A-Ⅰ via the pET-28a prokaryotic expression system. Mouse polyclonal antiserum was prepared by immunizing mice with purified recombinant protein. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titer of the polyclonal antiserum, and Western blot (WB) and indirect immunofluorescence assay (IFA) were used to determine its reactivity. Then, the polyclonal antiserum was further applied for subcellular localization analysis of chApo A-Ⅰ. Bioinformatic analysis revealed that the chApo A-Ⅰ protein contains signal peptide at 1-18 amino acids, which is composed of continuous alpha helix at the N-terminal. Homology analysis of amino acid sequences revealed that the chApo A-Ⅰ protein had the highest homology with turkey and the lowest homology with fish. The polyclonal antibody prepared using successfully expressed and purified recombinant protein His-chApo A-Ⅰ had an ELISA titer above 1×105 and specifically reacted with the eukaryotic expressed chApo A-Ⅰ protein in WB and IFA. Particularly, the antibody can recognize the Apo A-Ⅰ protein in chicken serum, but cannot cross-react with Apo A-Ⅰ proteins in the serums of mice, rabbits, cattle or pigs. This polyclonal antibody was further applied for subcellular localization analysis of full-length chApo A-Ⅰ (chApo A-Ⅰ-FL) and chApo A-Ⅰ without signal peptide (chApo A-Ⅰ-NS). Observed by confocal microscope, it was found that chApo A-Ⅰ-FL protein was mainly localized near the cell membrane, but chApo A-Ⅰ-NS protein was localized in the cytoplasm, and most of them were diffusely distributed. The specific polyclonal antibody and the results of subcellular localization of chApo A-Ⅰ provide a basis for further research on the biological function of Apo A-Ⅰ.
