Integrated analysis of the DNA methylome and RNA transcriptome during the development of skeletal muscle in Duroc pigs.

BACKGROUND: Skeletal muscle development plays a crucial role in yield and quality of pork; however, this process is influenced by various factors. In this study, we employed whole-genome bisulfite sequencing (WGBS) and transcriptome sequencing to comprehensively investigate the longissimus dorsi muscle (LDM), aiming to identify key genes that impact the growth and development of Duroc pigs with different average daily gains (ADGs). RESULTS: Eight pigs were selected and divided into two groups based on ADGs: H (774.89 g) group and L (658.77 g) group. Each pair of the H and L groups were half-siblings. The results of methylation sequencing revealed 2631 differentially methylated genes (DMGs) involved in metabolic processes, signalling, insulin secretion, and other biological activities. Furthermore, a joint analysis was conducted on these DMGs and the differentially expressed genes (DEGs) obtained from transcriptome sequencing of the same individual. This analysis identified 316 differentially methylated and differentially expressed genes (DMEGs), including 18 DMEGs in promoter regions and 294 DMEGs in gene body regions. Finally, LPAR1 and MEF2C were selected as candidate genes associated with muscle development. Bisulfite sequencing PCR (BSP) and quantitative real-time PCR (qRT-PCR) revealed that the promoter region of LPAR1 exhibited significantly lower methylation levels (P < 0.05) and greater expression levels (P < 0.05) in the H group than in the L group. Additionally, hypermethylation was observed in the gene body region of MEF2C, as was a low expression level, in the H group (P < 0.05). CONCLUSIONS: These results suggest that the differences in the ADGs of Duroc pigs fed the same diet may be influenced by the methylation levels and expression levels of genes related to skeletal muscle development.

Selection and identification of a precocious line of Eimeria intestinalis with enlarged oocysts and deletion of one generation of schizogony.

Rabbit coccidiosis is a common parasitic disease and responsible for enormous economic losses in the rabbit industry. Eimeria intestinalis, one of the highly pathogenic and common Eimeria species infecting rabbits, is considered as an indispensable species for the development of live oocyst vaccines against rabbit coccidiosis. In this study, we report the successful selection of a precocious line (EIP8) from a wild-type strain of E. intestinalis (WT) by successively collecting and propagating the early excreted progeny oocysts. The EIP8 line had a prepatent period of only 132 h compared to 204 h for the WT. Oocysts of EIP8 were notably different from those produced by the WT strain by their significantly larger size (mean length: 29.3 vs 27.6 µm and mean width 20.5 vs 19.8 µm). Examination of tissue sections prepared from EIP8-infected rabbits revealed that this precocious line undergoes only two generations of schizogony before differentiating into gametocytes by 120 h post-infection. In contrast, WT parasites undergo three generations of schizogony and gametocytes are present by 168 h post-infection. EIP8 multiplication capacity reduced by more than 35-fold and a concomitant decrease in pathogenicity was detected. Interestingly, immunization with 103 or 104 EIP8 oocysts provided sufficient protection against homologous challenge with wild-type parasites, as body weight gain of immunized and challenged rabbits was similar to that of untreated animals, as well as more than 90% reduction of oocyst output was detected in immunized and challenged animals when compared to unimmunized and challenged animals. Together, these results show that the EIP8 precocious line of E. intestinalis is an attenuated immunogenic strain and a suitable candidate for the development of a live vaccine against rabbit coccidiosis.

Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR.

PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists.

CDR3 analysis of TCR Vß repertoire of CD8⁺ T cells from chickens infected with Eimeria maxima.

CD8(+) T cells play a major role in the immune protection of host against the reinfection of Eimeria maxima, the most immunogenic species of eimerian parasites in chickens. To explore the dominant complementarity-determining regions 3 (CDR3) of CD8(+) T cell populations induced by the infection of this parasite, sequence analysis was performed in this study for CDR3 of CD8(+) T cells from E. maxima infected chickens. After 5 days post the third or forth infection, intraepithelial lymphocytes were isolated from the jejunum of bird. CD3(+)CD8(+) T cells were sorted and subjected to total RNA isolation and cDNA preparation. PCR amplification and cloning of the loci between Vß1 and Cß was conducted for the subsequent sequencing of CDR3 of T cell receptor (TCR). After the forth infection, 2 birds exhibited two same frequent TCR CDR3 sequences, i.e., AKQDWGTGGYSNMI and AGRVLNIQY; while the third bird showed two different frequent TCR CDR3 sequences, AKQGARGHTPLN and AKQDIEVRGPNTPLN. No frequent CDR3 sequence was detected from uninfected birds, though AGRVLNIQY was also found in two uninfected birds. Our result preliminarily demonstrates that frequent CDR3 sequences may exist in E. maxima immunized chickens, encouraging the mining of the immunodominant CD8(+) T cells against E. maxima infection.

Comparative analysis of productive performance and fattening efficiency of commercial pigs in China for two consecutive years.

THE PURPOSE OF THIS STUDY: (1) propose an evaluation indicator of the fattening efficiency of commercial pigs (Yorkshire × Landrace × Duroc)-fattening efficiency index (FEI). (2) Analyze the correlation to find the main productive factors affecting the FEI. (3) Compare and analyze the yearly/monthly/different piglets' sources of productive performance in 2020 and 2021. The data included 2592 commercial pig batches in 2020 and 3266 in 2021, with a total of 6,134,234 commercial pigs. Descriptive statistics and difference analysis were carried out on 16 productive factors of a whole year and single/multiple sources for two consecutive years. The same period difference between the monthly data and the annual average were also analyzed. The top six productive factors correlated with FEI were average daily gain (ADG) (0.8080), feed conversion rate (FCR) (- 0.7203), survival rate (SR) (0.6968), number of deaths (- 0.4103), feeding days (- 0.3748) and body weight (BW) of marketing pigs (0.3369). The overall productive performance in 2021 was lower than that in 2020, which was reflected in more piglet sources and a lower BW of piglets, more deaths, a lower SR, longer feeding days, a lower ADG, a higher FCR and a lower FEI. The productive performance of a single source was better than that of multiple ones. The contrastive results of monthly data in 2020 and 2021 showed significant differences in most factors except for the number of marketing pigs, the number of piglets and feed consumption. The monthly trend of 15 factors for two consecutive years revealed similar trends only in the month of piglets purchasing, number of piglets sources, number of deaths and ADG. Compared with the annual average, the ADG significantly increased in May. The FEI of multiple sources was markedly lower than that of a single source. FEI may be suitable for evaluating the fattening efficiency of commercial pigs. The annual and monthly productive performance and fattening efficiency in 2021 were significantly lower than those in 2020. Single source was represented better productive performance and fattening efficiency than multiple ones.

Effect of Risk-Focused Diversified Safety Management Mode in Patients with Major Artery Stent Implantation.

Background: Intracranial atherosclerotic stenosis (ICAS) causes a series of neurological symptoms, such as vertigo, impaired consciousness, limb weakness, ataxia, dysphagia, ocular motility disorders, and visual impairment. With the improvement of people's living standards, there are higher requirements for nursing care. Nursing, as an indispensable part of medical care, is closely related to achieving the goal of patient's safety and the overall quality of nurses, quality of care, and nursing management methods. Objective: To explore the effect of risk-centered diversified safety management in patients undergoing aortic stenting. Methods: Eighty patients with cerebral infarction were selected and treated with percutaneous transluminal angioplasty and stent implantation (PTAS). Then they were divided into a control group (40 cases) with routine monitoring and an experimental group (40 cases) with risk-focused intervention of a diversified safety management model according to the mode of care. Patient satisfaction and blood index test results were compared after the intervention. Results: Patients in the experimental group had 6 falls, 3 bed falls, 3 phlebitis, 4 tube slips, and 10 deep vein thrombosis, all significantly fewer than those in the control group. Thirty-eight patients in the experimental group expressed satisfaction with safe management, which was substantially better than the control group (P < 0.05). The levels of tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PA1-1), and von Willebrand factor (vWF) in the experimental group were (13.5 ± 1.3) ng/mL, (60.1 ± 9.9) ng/mL, and (2.1 ± 0.2), respectively, which were substantially lower than those in the control group ((14.6 ± 2.4) ng/mL, (64.2 ± 10.7) ng/mL, and (2.8 ± 0.3)), respectively (P < 0.05). Conclusion: The risk-centered diversified safety management model can effectively reduce the probability of adverse events in patients, improve patient satisfaction with nursing services, and promote faster postoperative recovery, which has clinical application value.

A new landscape of rabbit gut microbiota shaped by the infection of precocious parasites of Eimeria intestinalis.

Rabbit intestinal coccidiosis is caused by one or several Eimeria species, which cause intestinal damage and secondary bacterial infection. However, the impact of Eimeria infection on gut microbiota is much unknown. To evaluate the influence, we detected the feces flora of SPF rabbits infected with the 1 × 104 oocysts of E. intestinalis wild type (WT) and a precocious line (EIP8), a highly pathogenic species, by 16S rRNA sequencing. The microbiota of newly weaned rabbits post vaccination with low doses of EIP8 oocysts was also detected. In SPF rabbits, while Ruminococcaceae, Lachnospiraceae, and Bacteroidaceae were dominant families in all groups, EIP8 infection induced less changes in beta-diversity. In EIP8-infected rabbits, the intestinal flora whose abundance changed post infection accounted for less than 5.23 % of the entire flora. In comparison, it accounted for 27.18 % in WT group on d14 PI, while it was more than 20 % in diclazuril control group on d7 or d10 PI. The amount of fecal IgA and the abundance of IgA-production-related bacteria were similar in either EIP8 or WT infected rabbits. In the newly weaned rabbits, vaccination with EIP8 provided sufficient protection against challenge with WT parasites, as the body weight gain of vaccinated rabbits was similar to that of untreated animals, as well as more than 80 % reduction of oocyst output was detected when compared with unimmunized and challenged animals. Moreover, the vaccination had no significant impact on rabbit microbiota. Together, our findings suggested that the precocious line of E. intestinalis, compared with WT, induced a new fecal microbiota biodiversity in rabbits.

Rumen Microbiota Distribution Analyzed by High-Throughput Sequencing After Oral Doxycycline Administration in Beef Cattle.

The beef cattle rumen is a heterogenous microbial ecosystem that is necessary for the host to digest food and support growth. The importance of the rumen microbiota (RM) is also widely recognized for its critical roles in metabolism and immunity. The level of health is indicated by a dynamic RM distribution. We performed high-throughput sequencing of the bacterial 16S rRNA gene to compare microbial populations between rumens in beef cattle with or without doxycycline treatment to assess dynamic microbiotic shifts following antibiotic administration. The results of the operational taxonomic unit analysis and alpha and beta diversity calculations showed that doxycycline-treated beef cattle had lower species richness and bacterial diversity than those without doxycycline. Bacteroidetes was the predominant phylum in rumen samples without doxycycline, while Proteobacteria was the governing phylum in the presence of doxycycline. On the family level, the top three predominant populations in group qlqlwy (not treated with doxycycline) were Prevotellaceae, Lachnospiraceae, and Ruminococcaceae, compared to Xanthomonadaceae, Prevotellaceae, and Rikenellaceae in group qlhlwy (treated with doxycycline). At the genus level, the top predominant population in group qlqlwy was unidentified_Prevotellaceae. However, in group qlhlwy, the top predominant population was Stenotrophomonas. The results revealed significant RM differences in beef cattle with or without doxycycline. Oral doxycycline may induce RM composition differences, and bacterial richness may also influence corresponding changes that could guide antibiotic use in adult ruminants. This study is the first to assess microbiota distribution in beef cattle rumen after doxycycline administration.

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Taking banana continuous planting soil with high banana fusarium wilt disease incidence as a test site, we examined the effect of lime and ammonium carbonate fumigation coupled with bio-organic fertilizer on the suppression of banana fusarium wilt disease and the structure and composition of bacterial community, using real-time quantitative PCR and high-throughput sequencing. The results showed that the disease incidence was reduced by 13.3% and 21.7% in the treatments of LAOF (lime and ammonium carbonate fumigation coupled with organic fertilizer) and LABF (lime and ammonium carbonate fumigation coupled with bio-organic fertilizer), respectively, compared with OF (application of organic fertilizer without fumigation), while the copy number of Fusarium was decreased by 22.4% and 33.0%, respectively. Compared with non-fumigation treatment, lime and ammonium fumigation coupled with different fertilizer applications significantly reduced bacteria richness and diversity, with different community structure, while fumigation had a decisive effect on bacterial community composition. Bacterial richness and diversity of LABF were lower than those of other treatments, while microbial community structure was clearly disparate from other treatments. Compared with non-fumigation treatment, the relative abundance of Mizugakiibacter, Brucella, and Rhodanobacter were significantly improved in the fumigation coupled with different fertilization treatments. Those three genera in LABF were higher than those in LAOF, with significant differences for the relative abundances of Mizugakiibacter and Brucella. Therefore, fumigation combined with bio-organic fertilizer application could reduce the copy number of pathogen, alter soil bacterial community structure and stimulate beneficial bacteria in the resident soil, and thus reduce the occurrence of banana fusarium wilt.

High pathogenicity and strong immunogenicity of a Chinese isolate of Eimeria magna Pérard, 1925.

Coccidia infection of rabbits with one or several species of parasites of the genus Eimeria causes coccidiosis, a disease leading to huge economic losses in the rabbit industry. Eimeria magna, one of the causal agents of rabbit coccidiosis, was characterized as mildly pathogenic and moderately immunogenic in previous studies. In this study, we identified a Chinese isolate of E. magna by testing its biological features (oocyst morphology and size, prepatent time) and sequencing its internal transcribed spacer 1 (ITS-1) DNA fragment. This isolate is highly pathogenic; infection of rabbits with only 1×102 oocysts caused a 55% reduction in weight gain in 14days. In addition, immunization with 1×102 oocysts prevented body weight loss against re-infection with 5×104 oocysts, indicating the high immunogenicity of this isolate. Our study described the distinctive phenotype of the Chinese isolate of E. magna and contributed to the research of geographic variation of rabbit coccidia.

Transgenic Eimeria magna Pérard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.).

Rabbit coccidiosis causes great economic losses to world rabbitries. Little work has been done considering genetic manipulation on the etiological agents, rabbit Eimeria spp. In this study, we constructed a transgenic line of Eimeria magna (EmagER) expressing enhanced yellow fluorescent protein (EYFP) and red fluorescent protein (RFP) using regulatory sequences of Eimeria tenella and Toxoplasma gondii. We observed the life cycle of EmagER and confirmed that the transgenic parasites express exogenous proteins targeted to different cellular compartments throughout the entire life cycle. EYFP was expressed mainly in the nucleus and RFP both in the nucleus and cytoplasm. Then, coccidia-free, laboratory-reared 40-day-old rabbits were primarily infected with either EmagER or wild-type strain oocysts and challenged with the wild-type strain. EmagER showed similar reproductivity and immunogenicity to the wild-type strain. Finally, we examined the foreign protein-specific immune response elicited by EmagER. Rabbits were immunized with either transgenic or wild-type oocysts. Immune response against parasite-soluble antigen, EYFP and RFP in spleen, and mesenteric lymph nodes were detected by quantitative real-time PCR. The relative expression level of IFN-γ, IL-2, and TNF-α were higher in EmagER-immunized rabbits than wild-type parasites-immunized rabbits after stimulation with EYFP and RFP. Our study confirmed that a specific immune response was induced by the exogenous protein expressed by EmagER and favored future studies on application of transgenic rabbit coccidia as recombinant vaccine vectors.