Clearance of bacteria from lymph nodes in sheep immunized with Brucella suis S2 vaccine is associated with M1 macrophage activation.

Ovine brucellosis is a global zoonotic disease of sheep caused by Brucella melitensis, which inflicts a significant burden on human and animal health. Brucella suis strain S2 (B. suis S2) is a smooth live attenuated vaccine for the prevention of ovine brucellosis in China. However, no previous studies have assessed the immunogenicity of B. suis S2 vaccine after oral immunization in sheep. Here, we attempted to evaluate the ovine immune response over the course of B. suis S2 immunization and to identify in vivo predictors for vaccine development. Body temperature, serum Brucella antibodies, serum cytokines (IL-12p70 and interferon [IFN]-γ), and bacterial load in the mandibular lymph nodes (LN), superficial cervical LN, superficial inguinal LN, and spleen were investigated to determine the safety and efficacy of the vaccine. The abnormal body temperature of sheep occurred within 8 days post-infection (dpi). Brucella suis S2 persisted for a short time (< 21 dpi) in the mandibular LN. The highest level of IL-12p70 was observed at 9 dpi, whereas serum IFN-γ levels peaked at 12 dpi. Transcriptome analysis and quantitative reverse transcription PCR were performed to determine gene expression profiles in the mandibular LN of sheep. Antigen processing and presentation pathway was the dominant pathway related to the dataset. Our studies suggest that the immune response in ovine LN resembled type 1 immunity with the secretion of IL-12p70 and IFN-γ after B.suis S2 immunization and the vaccine may eliminate Brucella via stimulation of M1 macrophages through the course of Th cells.

Regulatory pathway analysis of coat color genes in Mongolian horses.

BACKGROUND: Studies on the molecular genetics of horse skin pigmentation have typically focused on very few genes and proteins. In this study, we used Illumina sequencing to determine the global gene expression profiles in horses with white-colored coats and those with black-colored coats, with the goal of identifying novel genes that could regulate horse coat color. RESULTS: Genes encoding ribosomal-associated proteins were highly expressed in horse skin. We found a total of 231 unigenes that were differentially expressed between horses with white coats and horses with black coats; 119 were down-regulated, and 112 were up-regulated. Many of the up-regulated genes in black horses, such as genes related to tyrosine metabolism, may directly regulate dark coat color. Keratin genes, MIA family genes, fatty acid-related genes, and melanoma-associated genes were also differentially regulated, which suggests that they may play important roles in coat color formation. CONCLUSIONS: These findings show that the transcription profiles from white and black horse skin provide useful information to understand the genetics underlying the control of skin melanin synthesis in horses, which may enhance our knowledge of human skin diseases, such as melanoma and albinism.

Comparison of Fecal Microbiota of Mongolian and Thoroughbred Horses by High-throughput Sequencing of the V4 Region of the 16S rRNA Gene.

The hindgut of horses is an anaerobic fermentative chamber for a complex and dynamic microbial population, which plays a critical role in health and energy requirements. Research on the gut microbiota of Mongolian horses has not been reported until now as far as we know. Mongolian horse is a major local breed in China. We performed high-throughput sequencing of the 16S rRNA genes V4 hypervariable regions from gut fecal material to characterize the gut microbiota of Mongolian horses and compare them to the microbiota in Thoroughbred horses. Fourteen Mongolian and 19 Thoroughbred horses were used in the study. A total of 593,678 sequence reads were obtained from 33 samples analyzed, which were found to belong to 16 phyla and 75 genera. The bacterial community compositions were similar for the two breeds. Firmicutes (56% in Mongolian horses and 53% in Thoroughbred horses) and Bacteroidetes (33% and 32% respectively) were the most abundant and predominant phyla followed by Spirochaete, Verrucomicrobia, Proteobacteria, and Fibrobacteres. Of these 16 phyla, five (Synergistetes, Planctomycetes, Proteobacteria, TM7, and Chloroflexi) were significantly different (p<0.05) between the two breeds. At the genus level, Treponema was the most abundant genus (43% in Mongolian horses vs 29% in Thoroughbred horses), followed by Ruminococcus, Roseburia, Pseudobutyrivibrio, and Anaeroplasma, which were detected in higher distribution proportion in Mongolian horses than in Thoroughbred horses. In contrast, Oscillibacter, Fibrobacter, Methanocorpusculum, and Succinivibrio levels were lower in Mongolian horses. Among 75 genera, 30 genera were significantly different (p<0.05) between the two breeds. We found that the environment was one of very important factors that influenced horse gut microbiota. These findings provide novel information about the gut microbiota of Mongolian horses and a foundation for future investigations of gut bacterial factors that may influence the development and progression of gastrointestinal disease in horses.

Downregulation of Three Novel miRNAs in the Lymph Nodes of Sheep Immunized With the Brucella suis Strain 2 Vaccine.

Ovine and caprine brucellosis, both caused by Brucella melitensis, lead to substantial economic losses in the animal industry and health problems in human populations. Brucella suis strain 2 (B.suis S2), as a live attenuated vaccine, is used extensively in China to prevent brucellosis. It has been proven that microRNA (miRNAs) are involved in the immunopathogenesis of brucellosis; however, the miRNA-driven mechanism of immune response to B.suis S2 in vivo remains unknown. To determine which new miRNAs are involved in the host immune response to B.suis S2 and elucidate the function of these miRNAs, we performed a comprehensive analysis of miRNA expression profiles in sheep immunized with B.suis S2 using the high-throughput sequencing approach. The submandibular lymphatic nodes from sheep seropositive for Brucella were collected at 7, 14, 21, 30, 60 and 90 days post-immunization. MiRNA sequencing analysis revealed that 282 differentially expressed miRNAs (|log2 fold-change |>0.5 and p < 0.05) were significantly enriched in the immune pathways, including the NF-kappa B signaling pathway, B cell receptor signaling pathway, p53 signaling pathway and complement and coagulation cascades. Increasing the threshold to |log2 fold change|>1 and p < 0.01 revealed 48 differentially expressed miRNAs, 31 of which were novel miRNAs. Thirteen of these novel miRNAs, which were differentially expressed for at least two time points, were detected via RT-qPCR assays. The novel_229, novel_609, novel_973 and oar-miR-181a assessed by RT-qPCR were detectable and consistent with the expression patterns obtained by miRNA sequencing. Functional analyses of these miRNAs demonstrated that their target genes participated in the immune response pathways, including the innate and adaptive immunity pathways. The immune-related target genes of novel_229 included ENSOARG00000000649 and TMED1, as well as LCN2, PDPK1 and LPO were novel_609 target genes. The immune-related target genes of novel_973 included C6orf58, SPPL3, BPIFB1, ENSOARG00000021083, MPTX1, CCL28, FGB, IDO1, OLR1 and ENSOARG00000020393. The immune-related target genes of oar-miR-181a included ENSOARG00000002722, ARHGEF2, MFAP4 and DOK2. These results will deepen our understanding of the host miRNA-driven defense mechanism in sheep immunized with B.suis S2 vaccine, and provide the valuable information for optimizing vaccines and developing molecular diagnostic targets.

[Effects of Kit gene on coat depigmentation in white horses].

Coat color of horse is an important basis for both species identification and individual recognition and is also one of the important references traits for breeding. Therefore, the research on the mechanism of coat fading has become an important part of horses' coat color study. It has been found that the white phenotype is closely related to the mutation of kit gene, which is located on chromosome 3. Investigated results showed that the formation of the epidermal melanoblast and melanin relies on the expression of kit gene, which determines the presence of white phenotype. Nevertheless, studies results have shown that the mutation of kit gene in the white horse exhibited significant differences among species. Horses that the coat color completely faded are very rare and are found occasionally in a few species. However, a larger number of horses that coat color completely faded, called Mongolian white horse, are found in West Ujimqin , Xilin Gol League, Inner Mongolia. Therefore, genetic mechanism of color fading in Mongolian white horses is still not clear. No typical mutations have been observed in 21 exons of kit gene in Mongolian white horse. This paper summarized recent international studies on molecular mechanism of color fading and tried to lay the foundation for the study of formation mechanism of Mongolian white horse. The aim of this review is to provide some valuable references to horses coat color research and breeding.

Radiosynthesis and evaluation of N5-(2-18F-fluoropropanyl) ornithine as a potential agent for tumor PET imaging.

OBJECTIVE: Studies have confirmed that tumorigenesis is related to an imbalance of polyamine metabolism and over-expression of oncogenes resulting in the up-regulation of ornithine decarboxylase (ODC, the first rate-limiting enzyme for regulating intracellular polyamines biosynthesis), which has become a target for anti-tumor therapy. In this study, an ornithine derivative, N5-(2-[18F]fluoropropionyl) ornithine (N5-[18F]FPO), has been prepared and its potential utility for tumor PET imaging evaluated. METHODS: N5-[18F]FPO was successfully prepared via a nucleophilic fluorination reaction and a subsequent efficient deprotection step. The in vitro and in vivo stability were determined by HPLC conducted in fetal bovine serum, saline and rat urine. Cellular uptake studies were conducted in HepG2 cells and the biodistribution and micro-PET/CT imaging performed in normal ICR mice and three tumor-bearing mice models, respectively. RESULTS: Total synthesis time of N5-[18F]FPO was about 80 min with a radiochemical yield of 15% ± 6% (uncorrected, based on 18F-, n = 6) and a high radiochemical stability can be seen in vitro and vivo. The N5-[18F]FPO exhibited fast uptake in HepG2 cells and the cellular uptake ability of N5-[18F]FPO can be inhibited by L-ornithine and DFMO, which indicated that the transport pathway of N5-[18F]FPO is similar to that of L-ornithine, interacting with ODC after being transported into the cell. The biodistribution and micro-PET/CT images demonstrate that N5-[18F]FPO was excreted by the urinary system, and excellent tumor visualization with high tumor-to-background ratios can be observed in the three tumor-bearing mice models studied. CONCLUSION: All the above results suggest that N5-[18F]FPO has the potential to be a novel radiotracer for imaging ODC expression in solid tumors.

Synthesis of N-(6-[18F]Fluoropyridin-3-yl)glycine as a potential renal PET agent.

OBJECTIVE: Given the requirements of high sensitivity and spatial resolution, the development of new positron emission tomography (PET) agents is required for PET renography. The objective of this study was to investigate a new fluorine-18 labeled hippurate analogue of picolinamide, N-(6-[18F]Fluoropyridin-3-yl)glycine, as a new renal PET agent for evaluating renal function. METHODS: N-(6-[18F]Fluoropyridin-3-yl)glycine was prepared via a two-step reaction, including the nucleophilic substitution reaction of Br with 18F using methyl 2-(6-bromonicotinamido)acetate as a precursor followed the hydrolysis with sodium hydroxide and purification by preparative-HPLC. The in vitro and in vivo stability were determined using HPLC, and the plasma protein binding (PPB) and erythrocyte uptake of N-(6-[18F]Fluoropyridin-3-yl)glycine were determined using blood collected from healthy rats at 5 min post-injection. Biodistribution and dynamic micro-PET/CT imaging studies were conducted in healthy rats. RESULTS: N-(6-[18F]Fluoropyridin-3-yl)glycine was prepared within 45 min with an uncorrected radiochemical yield of 24.5 ±â€¯6.7% (n = 6, based on [18F]F-) and a radiochemical purity of >98%. N-(6-[18F]Fluoropyridin-3-yl)glycine demonstrated good stability both in vitro and in vivo. The results of the biodistribution and dynamic micro-PET/CT imaging studies in normal rats indicated that N-(6-[18F]Fluoropyridin-3-yl)glycine was rapidly and exclusively excreted via the renal-urinary pathway. CONCLUSION: N-(6-[18F]Fluoropyridin-3-yl)glycine is has been shown to be a promising renal PET agent and warrants further evaluation of renal function.

Donkey genome and insight into the imprinting of fast karyotype evolution.

The donkey, like the horse, is a promising model for exploring karyotypic instability. We report the de novo whole-genome assemblies of the donkey and the Asiatic wild ass. Our results reflect the distinct characteristics of donkeys, including more effective energy metabolism and better immunity than horses. The donkey shows a steady demographic trajectory. We detected abundant satellite sequences in some inactive centromere regions but not in neocentromere regions, while ribosomal RNAs frequently emerged in neocentromere regions but not in the obsolete centromere regions. Expanded miRNA families and five newly discovered miRNA target genes involved in meiosis may be associated with fast karyotype evolution. APC/C, controlling sister chromatid segregation, cytokinesis, and the establishment of the G1 cell cycle phase were identified by analysis of miRNA targets and rapidly evolving genes.

Analysis of horse genomes provides insight into the diversification and adaptive evolution of karyotype.

Karyotypic diversification is more prominent in Equus species than in other mammals. Here, using next generation sequencing technology, we generated and de novo assembled quality genomes sequences for a male wild horse (Przewalski's horse) and a male domestic horse (Mongolian horse), with about 93-fold and 91-fold coverage, respectively. Portion of Y chromosome from wild horse assemblies (3 M bp) and Mongolian horse (2 M bp) were also sequenced and de novo assembled. We confirmed a Robertsonian translocation event through the wild horse's chromosomes 23 and 24, which contained sequences that were highly homologous with those on the domestic horse's chromosome 5. The four main types of rearrangement, insertion of unknown origin, inserted duplication, inversion, and relocation, are not evenly distributed on all the chromosomes, and some chromosomes, such as the X chromosome, contain more rearrangements than others, and the number of inversions is far less than the number of insertions and relocations in the horse genome. Furthermore, we discovered the percentages of LINE_L1 and LTR_ERV1 are significantly increased in rearrangement regions. The analysis results of the two representative Equus species genomes improved our knowledge of Equus chromosome rearrangement and karyotype evolution.