BackgroundHypervirulent Klebsiella pneumoniae (hvKp) is a critical pathogen that causes highly lethal invasive infections with its virulence linked to that of aerobactin, the core siderophore system of hvKp.MethodsHere, 20 porcine-derived K. pneumoniae strains were subjected to capsular genotyping and the virulence of the strains was evaluated through mouse lethality assays. Whole-genome sequencing and analysis was performed on two K2 serotype strains (including highly and weakly virulent strains), and the differential virulence genes were analyzed. The virulence-related gene iucC, identified in the hypervirulent K2 serotype strain KP10, was knocked out using homologous recombination to preliminarily explore its function in K. pneumoniae.ResultsIn this study, a highly virulent K2-type strain KP10 and a low virulent K2-type strain KP6 were screened from 20 strains of porcine K. pneumoniae. The results of the genome and virulence gene analyses revealed that compared with KP6, the highly virulent strain KP10 specifically encodes the iucC gene. The △ iucC mutant of the high-virulence strain KP10 exhibited significantly reduced biofilm formation, decreased siderophore production, moderate serum sensitivity, and attenuated virulence. Furthermore, the mutant displayed decreased adhesion to IPEC-J2 cells, reduced cytotoxicity, and decreased cell mortality. These results revealed that iucC is a major contributor to virulence in the KP10 hvKp strain. Notably, iucC genes were detected in the genomes of K. pneumoniae from humans and six different animal and environmental sources, and some iucC genes were shared in K. pneumoniae from these sources.ConclusionOverall, this work provides a crucial theoretical foundation for elucidating iucC function and the pathogenesis of K. pneumoniae. Its widespread presence in different hosts and environments considerably increases the risk of cross species transmission and public health events.

Streptococcus equi subsp. equi causes the equine respiratory disease ‘strangles’, which is highly contagious, debilitating and costly to the equine industry. S. equi emerged from the ancestral Streptococcus equi subsp. zooepidemicus and continues to evolve and disseminate globally. Previous work has shown that there was a global population replacement around the beginning of the twentieth century, obscuring the early genetic events in this emergence. Here, we have used large-scale genomic analysis of S. equi and its ancestor S. zooepidemicus to identify evolutionary events, leading to the successful expansion of S. equi . One thousand two hundred one whole-genome sequences of S. equi were recovered from clinical samples or from data available in public databases. Seventy-four whole-genome sequences representative of the diversity of S. zooepidemicus were used to compare the gene content and examine the evolutionary emergence of S. equi . A dated Bayesian phylogeny was constructed, and ancestral state reconstruction was used to determine the order and timing of gene gain and loss events between the different species and between different S. equi lineages. Additionally, a newly developed framework was used to investigate the fitness of different S. equi lineages. We identified a novel S. equi lineage, comprising isolates from donkeys in Chinese farms, which diverged nearly 300 years ago, after the emergence of S. equi from S. zooepidemicus , but before the global sweep. Ancestral state reconstruction demonstrated that phage-encoded virulence factors slaA , seeL and seeM were acquired by the global S. equi after the divergence of the basal donkey lineage. We identified the equibactin locus in both S. equi populations, but not S. zooepidemicus , reinforcing its role as a key S. equi virulence mechanism involved in its initial emergence. Evidence of a further population sweep beginning in the early 2000s was detected in the UK. This clade now accounts for more than 80% of identified UK cases since 2016. Several sub-lineages demonstrated increased fitness, within which we identified the acquisition of a new, fifth prophage containing additional toxin genes. We definitively show that acquisition of the equibactin locus was a major determinant in S. equi becoming an equid-exclusive pathogen, but that other virulence factors were fixed by the population sweep at the beginning of the twentieth century. Evidence of a secondary population sweep in the UK and acquisition of further advantageous genes implies that S. equi is continuing to adapt, and therefore, continued investigations are required to determine further risks to the equine industry.

High-quality colostrum is crucial in preventing hypogammaglobulinemia and infectious diseases in foals. This study aims to estimate the suitability of a Brix refractometer for estimating IgG concentration. To estimate the reliability of using the refraction method (Brix refractometer) to assess colostrum quality in Dezhou donkeys by comparing its performance with that of the radial immunodiffusion method. Colostrum samples (n=69) were collected from 23 Dezhou jennies at 0, 6, and 12 hours after birth. Regression analysis and one-way analysis of variance (ANOVA) were carried out on the collected data. The results showed strong correlations between the two methods (P<0.01). Regression analysis revealed a statistically significant relationship between the IgG concentration and Brix value (R-2=0.9177). By using the regression equation between the two methods, the colostrum of Dezhou donkeys could be classified into four categories: excellent, good, fair, and poor quality, based on Brix values greater than 17.96, between 15.03 and 17.96, between 12.88 and 15.03, and less than 12.88%, respectively. These results suggested that the refraction method can be a reliable and efficient alternative to estimate colostrum quality in Dezhou donkeys.

This paper represents the fundamental report of the survey of genome-wide changes of four Chinese indigenous donkey breeds, Dezhou (DZ), Guangling (GL), North China (NC), and Shandong Little donkey (SDL), and the findings will prove usefully for identification of biomarkers that perhaps predict or characterize the growth and coat color patterns. Three genomic regions in CYP3A12, TUBGCP5, and GSTA1 genes, were identified as putative selective sweeps in all researched donkey populations. The loci of candidate genes that may have contributed to the phenotypes in body size (ACSL4, MSI2, ADRA1B, and CDKL5) and coat color patterns (KITLG and TBX3) in donkey populations would be found in underlying strong selection signatures when compared between large and small donkey types, and between different coat colors. The results of the phylogenetic analysis, FST, and principal component analysis (PCA) supported that each population cannot clearly deviate from each other, showing no obvious population structure. We can conclude from the population history that the formation processes between DZS and NC, GL, and SDL are completely different. The genetic variants discovered here provide a rich resource to help identify potential genomic markers and their associated molecular mechanisms that impact economically important traits for Chinese donkey breeding programs.

高脂血症(hyperlipidemia)是驴规模化繁育场最常见的疾病之一,具有发病率高、死亡率高、早期诊断困难等特点,严重危害中国驴产业的健康发展.驴的酮体生成途径不发达,加之天生的胰岛素抵抗等多因素共同作用使得驴更易患高脂血症.这种疾病是由能量负平衡引发的脂肪组织过度动员,可分为由应激、肥胖、妊娠、泌乳和遗传等因素导致的原发性高脂血症,以及由各种导致机体能量负平衡的全身性疾病所引起的继发性高脂血症.其病因和发生机制主要包括血液脂质堆积、组织对胰岛素抵抗、原发性疾病、肠道菌群紊乱和遗传效应等.该病可通过测定血清脂质浓度结合其临床表现进行诊断.随着代谢组学的发展,也可通过找寻相关的小分子生物标志物,结合血清的甘油三酯浓度对高脂血症做出更准确的早期诊断.笔者从病因及发生机制、临床诊断方法方面对驴高脂血症进行综述,并展望了驴高脂血症未来的研究方向,以推动驴高脂血症的临床诊疗和抗病育种技术的发展.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the most common pathogens of endometritis in intensive donkey farms. In order to investigate the molecular epidemiological characteristics of S. zooepidemicus isolates associated with endometritis in intensive donkey breeding farms in China, isolates (n=40) from 8 small and medium-sized breeding farms were selected for multilocus sequence typing analysis. Results revealed that 4 sequence types (STs) were identified, ST360, ST380, ST413, and a novel genotype ST-novel, and these were genetically distant from each other, which suggested that S. zooepidemicus strains caused endometritis in intensive donkey farms possessed high genetic diversity and were lack of genotype specificity. The result of present study contributed to the understanding of the molecular epidemiological characteristics of S. zooepidemicus isolates

Background In contrast to horses, the only evidence suggesting gastrointestinal disease in neonatal donkeys is associated with Group A rotaviruses (RVAs) is the detection of viral antigens by ELISA in just 1 of 82 symptomatic donkey foals. No additional, more comprehensive investigations have been conducted, and RVAs if circulating in donkey populations have not been molecularly characterised. Objectives To investigate if RVAs are associated with an outbreak of severe enteritis in neonatal donkeys and if associated determine the genotype(s) along with the phylogenetic relationship to RVA strains circulating in horses. Study design Cross-sectional. Methods RT-PCR-based techniques were used for RVA diagnosis and gene amplification. Statistical significance was determined by Chi-square and Fisher's exact two-sided tests. Genotyping was performed by RotaC and phylogenetic analysis by neighbour joining. Results In 2019, acute enteritis occurred in 119 of 206 donkey foals (<= 4 months) at two intensive donkey farms in the Shandong province of China. The highest morbidity (68.1%), mortality (29.5%) and fatality levels (45.5%) occurred in foals in the 30-89 day, 30-59 day and 0-29 day age groups respectively. RVA gene sequences were detected in 107 (89.9%) of the symptomatic individuals while further analysis demonstrated the outbreak was associated with the same G3P[12] RVA strain designated RVA/Donkey-wt/CHN/Don01/2019/G3P[12]. Although the VP4 gene of Don01 exhibited close phylogenetic relationships with equivalent RVA sequences commonly circulating in horses, encoding VP7 was more closely associated with sequences isolated from bats suggesting this new donkey strain arose via an intergenogroup reassortment event. Main limitations Actual prevalence not determined because Valuable new information about the molecular epidemiology of rotaviruses in different equid species is provided by isolation and molecular characterisation of a novel RVA strain from neonatal donkeys.

In contrast to horses, the only evidence suggesting gastrointestinal disease in neonatal donkeys is associated with Group A rotaviruses (RVAs) is the detection of viral antigens by ELISA in just 1 of 82 symptomatic donkey foals. No additional, more comprehensive investigations have been conducted, and RVAs if circulating in donkey populations have not been molecularly characterised. To investigate if RVAs are associated with an outbreak of severe enteritis in neonatal donkeys and if associated determine the genotype(s) along with the phylogenetic relationship to RVA strains circulating in horses. Cross-sectional. RT-PCR-based techniques were used for RVA diagnosis and gene amplification. Statistical significance was determined by Chi-square and Fisher's exact two-sided tests. Genotyping was performed by RotaC and phylogenetic analysis by neighbour joining. In 2019, acute enteritis occurred in 119 of 206 donkey foals (≤4 months) at two intensive donkey farms in the Shandong province of China. The highest morbidity (68.1%), mortality (29.5%) and fatality levels (45.5%) occurred in foals in the 30-89 day, 30-59 day and 0-29 day age groups respectively. RVA gene sequences were detected in 107 (89.9%) of the symptomatic individuals while further analysis demonstrated the outbreak was associated with the same G3P[12] RVA strain designated RVA/Donkey-wt/CHN/Don01/2019/G3P[12]. Although the VP4 gene of Don01 exhibited close phylogenetic relationships with equivalent RVA sequences commonly circulating in horses, encoding VP7 was more closely associated with sequences isolated from bats suggesting this new donkey strain arose via an intergenogroup reassortment event. Actual prevalence not determined because <7% of asymptomatic donkey foals were included in this study. The complete genomic sequence of RVA/Donkey-wt/CHN/Don01/2019/G3P[12] remains to be determined. Valuable new information about the molecular epidemiology of rotaviruses in different equid species is provided by isolation and molecular characterisation of a novel RVA strain from neonatal donkeys.

Donkeys' gut microbe is critical for their health and adaptation to the environment. Little research has been conducted on the donkey gut microbiome compared with other domestic animals. The Tibetan Plateau is an extreme environment. In this study, 6 Qinghai donkeys (QH) from the Tibetan Plateau and 6 Dezhou donkeys (DZ) were investigated, and the contents of 4 parts-stomach, small intestine, cecum, and rectum-were collected. 16S rRNA sequencing and metagenomic sequencing were used to analyze the composition and diversity of gut microbial communities in donkeys. The results showed that the flora diversity and richness of the hindgut were significantly higher than those of the foregut (p < 0.01), with no sex differences, and the community structure and composition of the same or adjacent regions (stomach, small intestine, cecum, and rectum) were similar. Besides, the flora diversity and richness of QH on the Tibetan Plateau were significantly higher than those of DZ (p < 0.05). The major pathways associated with QH were signal transduction mechanisms and carbohydrate transport and metabolism, and Bacteroidales were the major contributors to these functions. Our study provides novel insights into the contribution of microbiomes to the adaptive evolution of donkeys.

为研究青海毛驴的肠道微生物优势菌群,本实验选取来自成年、健康的青海省青海毛驴和山东省德州驴各6头(公母各半),分别采集胃、小肠、盲肠和直肠内容物样品.利用16S rRNA高通量测序及生物信息学分析青海毛驴和德州驴不同部位的肠道微生物细菌群落的组成和多样性.结果显示:青海毛驴的菌群多样性和丰富度高于德州驴(P≤0.05),后肠的菌群多样性和丰富度均高于前肠(P≤0.01),且在相似的地理环境下和来自相同/相邻区域(胃和小肠,盲肠和直肠)的微生物,其群落结构和组成更相似.

Biofilm formation is a fundamental part of life cycles of bacteria which affects various aspects of bacterial-host interactions including the development of drug resistance and chronic infections. In clinical settings, biofilm-related infections are becoming increasingly difficult to treat due to tolerance to antibiotics. Bacterial biofilm formation is regulated by different external and internal factors, among which quorum sensing (QS) signals and nucleotide-based second messengers play important roles. In recent years, different kinds of anti-biofilm agents have been discovered, among which are the Chinese herbal medicines (CHMs). CHMs or traditional Chinese medicines have long been utilized to combat various diseases around the world and many of them have the ability to inhibit, impair or decrease bacterial biofilm formation either through regulation of bacterial QS system or nucleotide-based second messengers. In this review, we describe the research progresses of different chemical classes of CHMs on the regulation of bacterial biofilm formation. Though the molecular mechanisms on the regulation of bacterial biofilm formation by CHMs have not been fully understood and there are still a lot of work that need to be performed, these studies contribute to the development of effective biofilm inhibitors and will provide a novel treatment strategy to control biofilm-related infections.

应激是动物机体在受到各种内外因素刺激时所出现的一系列病理反应,动物在应激状态下,体内会迅速产生大量活性氧自由基(ROS),当过量的自由基无法及时清除时,就会引起动物的氧化损伤,甚至导致应激性疾病的发生.过氧化氢酶(CAT)可以清除体内的ROS,预防或改善动物的应激反应,研究表明它不仅具有保护动物肠道健康、抑制炎症反应、增强动物机体免疫力等生理功能,而且在减少细胞损伤、抑制细胞凋亡、预防肿瘤发生等方面也有着重要的潜在应用价值.

Phage therapy is a promising alternative therapy for the treatment of E. coli infection. Although the total number of phages on the earth is as high as 10 31 , the reported phages and thoroughly studied are very limited. Therefore, the continuous discovery of new phages and in-depth research will provide materials for the wide application of phage therapy in the future. In this study, a novel E. coli phage vB_EcoM_011D4 was isolated from sewage samples, and the biological characteristics were studied. Electron microscopy and homology analysis results showed that vB_EcoM_011D4 belongs to the family Myoviridae. One-step growth curve showing the latent period of vB_EcoM_011D4 was 10 min, with the burst size of 115 PFU/cell. Additionally, Phage vB_EcoM_011D4 was highly stabled under different temperatures (range 4 – 70 ℃) and pH conditions (range 6 – 10). At the same time, its genome was subjected to high-throughput sequencing and compared with the reported phages. The results of high-throughput sequencing assembly showed that vB_EcoM_011D4 is a linear, double-stranded DNA virus containing 163764 bp, with an average GC content of 40.50%, and a total of 273 open reading frames (ORFs). Genomic comparison analysis revealed that most of the ORFs were similar to Enterobacteria phage Phi1 and RB49. However, ORF147 and ORF148 putative DNA methylase family protein is less than 67% homology with already published phages. In addition, the phylogenetic analysis of terminates large subunit showed that it belongs to a new branch and shows less than 50 similarities to reported phages. There is no lysogenic, toxin or antibiotic-resistant related gene was found in the genome of vB_EcoM_011D4. In summary, vB_EcoM_011D4 is a newly discovered phage, which can be further studied for elucidating the phage diversity and it is benefits for the wide application of phage therapy.

从发生腺疫的青海毛驴中采集样品进行染色镜检、分离纯化、PCR鉴定,并对纯化后的菌株进行药敏试验.研究结果表明:美兰染色结果观察到长链状球菌;5％绵羊血平板培养出现透明的露珠样小菌落,且呈现β型溶血;PCR鉴定为马链球菌马亚种;药敏试验表明马链球菌马亚种对β内酰胺类抗生素敏感.

S . Abortusequi is an important pathogen that can induce abortions in mares. Although S . Abortusequi has been well controlled in Europe and the United States due to strict breeding and health policies, it is still widespread in African and Asian countries and has proven difficult to control. In China, abortions caused by S . Abortusequi have also been reported in donkeys. So far, there is no commercial vaccine. Thus, exploiting alternative efficient and safe strategies to control S . Abortusequi infection is essential. In this study, a new lytic phage, P IZ SAE-01E2, infecting S . Abortusequi was isolated, and the characteristics of P IZ SAE-01E2 indicated that it has the potential for use in phage therapy. A single intraperitoneal inoculation of P IZ SAE-01E2 before or after S . Abortusequi challenge provided effective protection to all pregnant mice. Thus, P IZ SAE-01E2 showed the potential to block abortions induced by S . Abortusequi in vivo .

Endometritis has recently become one of the most important infectious diseases in donkey breeding farms in China. However, so far, no research has been conducted to confirm the causative agents. In the present study, one breeding farm was selected as a research model for isolation and identification of causative agents and epidemiological investigation. Streptococcus equi subspecies zooepidemicus was isolated from the uterus of symptomatic and asymptomatic donkeys and identified by PCR and MALDI-TOF MS analysis. On the farm, the infection rate was 57.1%, which indicated that endometritis is a severe problem and needs to pay much attention to donkey breeding farms. Moreover, female donkeys without mating experience were all negative which suggested the transmitting route might be artificial insemination or related handlings. This study will lead to a great understanding of the causative agent and epidemiological characteristics for endometritis in intensive donkey breeding farms

The application of phages against the increased reported drug resistant Escherichia coli is one of the promising alternative therapeutic options. The total number of phages on the earth are more than 10(31), but the phages that have been isolated and studied are limited. Hence, discovering of new phage and uncovering its characteristics will provide materials for extensive use of phage therapy in the future. In this study, a novel E. coli phage named vB_EcoS_W011D was isolated and the characteristics and genome were explored. The typical morphology of vB_EcoS_W011D is comprised of an icosahedral head and a constricted flexible rolled up tail, revealing that it is the genus TLS virus of Tunavirinae subfamily. One-step growth curve showing the eclipse and latent period of vB_EcoS_W011D was 5 min and 10 min, respectively, with the burst size of 115 PFU/cell. The genome of vB_EcoS_W011D is double-stranded consisting of 49,847 bp with 46.24% of G+C contents and shows <= 77% similarities (with 38% query coverage) to other reported phages. A total 85 putative ORFs were identified. Of which, 43 predicted ORFs had significant homology with other phage proteins of known functions. A putative Zonula occludes toxin was found in its genome. In addition, a clear difference was revealed on the phylogenetic analysis of it terminates large subunit and capsid protein. In conclusion, our study clearly indicates that vB_EcoS_W011D is a newly discovered E. coli phage that could be further investigated to elucidate phage variety and evolutionary relationship between bacteria and phages. (C) 2019 PVJ. All rights reserved

研究开放式采精对种公马采精准备时间、爬跨次数、射精时间、射精量、精子密度、精子活率和24h保鲜精精子活率的影响.6匹种公马分为试验组和对照组,试验组采用开放式采精,对照组采用普通密闭采精,测定上述指标并进行统计学分析.结果 表明,种公马准备时间、爬跨次数和射精时间试验组均显著高于对照组,第二周试验组均极显著低于第一周试验组;试验组精子密度极显著高于对照组(P＜0.01);精子活率各组无显著差异;24h精子活率试验组极显著高于对照组(P＜0.01),而两周试验组无显著差异(P＞0.05).种公马可适应开放式采精且采集的精液保鲜精活率较高.

Bovine mastitis is an inflammatory response mainly caused by Staphylococcus aureus. Lysin is a cell wall hydrolase encoded and synthesized by a bacteriophage, which can kill specific Gram-positive bacteria. In this study, phage lysin “LysGH15” is used to treat the mice mastitis caused by S. aureus. The purified lysGH15 showed strong bactericidal activity in vitro. When treated with 25μg/mL of the LysGH15, the bacterial counts of S. aureus dropped approximately 5 log units within 10 min. In the in vivo experiments, the administration of LysGH15 significantly (P<0.05) reduced the colonies of S. aureus and alleviated damage to the breast tissue. Also, the levels of IL-6 and TNF-α in breast tissue were significantly decreased. It indicates that the LysGH15 can effectively treat the murine mastitis caused by S. aureus. This study demonstrated the potential of LysGH15 as an alternative to antibiotics for treating bovine mastitis caused by S. aureus