Virulence, cytotoxic and inflammatory activities of Vibrio anguillarum and Aeromonas salmonicida isolated from cultivated salmonid fish in Sweden

Extracellular products in culture filtrates of Aeromonas salmonicida subsp. achromogenes and Vibrio anguillarum isolated from infected fish have been shown to possess skin inflammatory factor. The extracellular products from Vibrio anguillarum were cytotoxic in HeLa and CHO cells. In addition to the skin lesions, the culture filtrates of V. anguillarum caused necrotic reaction on the rabbit skin. Five of 6 strains of V. anguillarum were lethal to mice after intraperitoneal administration of 3×10⁷ CFU. Only 1 strain of 5 A. salmonicida subsp. achromogenes produced extracellular products which elicited cytotoxic effects in the CHO cells. None of the A. salmonicida subsp. achromogenes strains were lethal to mice. The cytotoxins were inactivated when heated at 65°C for 30 min. The results indicate that the thermolabile exotoxins are non-enterotoxic since they failed to stimulate fluid accumulation in the rabbit ileal loop and did not cause elongation of the CHO cells. The rounding off of CHO cells, as well as of HeLa cells indicate that the exotoxins may play an important role in fish diseases.     

杨梅癌肿病病原菌的侵染途径及肿瘤的形态发生初报

试验说明杨梅癌肿病病原细菌也可从叶痕或皮孔侵入茎,在薄壁细胞内大量繁殖并继续向茎皮层侵染,皮层薄壁细胞受到细菌分泌物的刺激,细胞分裂,使皮层细胞数目增多,向外突出形成肿瘤,肿瘤内有大量薄壁细胞及排列不规则的输导组织;后者与茎内的维管组织相连,结果使寄主体内的部分物质物质运往病部,消耗于肿瘤的生长,枝梢得不到充足的水分和养料,可能是造成肿瘤以上枝梢枯死的原因之一。     

Evaluation of Therapeutic Effect of Porcine β-defensin-2 on Streptococcus suis Infection

Defensin is a small cationic peptide widely distributed in animals and plants. It is an important component of the innate immune defense system of mammals. It has a broad spectrum of antibacterial, antiviral and antifungal activities. Porcine β-defensin-2 (PBD-2) is one of the natural defensins expressed in pigs and has good antibacterial activity in vitro. The purpose of this study is to evaluate the therapeutic effect of PBD-2 in the treatment of Streptococcus suis infection in animals and to provide more insights for evaluating the value of PBD-2 as a therapeutic drug. Firstly, the bactericidal activity of PBD-2 against S. suis clinical isolate SC-19 was measured in vitro, and the cytotoxicity of PBD-2 was tested on mouse-derived macrophages (RAW264.7). Subsequently, C57 female mice aged 4-6 weeks and weighing between 18-22 g infected with S. suis SC-19 were treated with PBD-2 or PBS (n ≥ 6). The survival rate of mice, and the bacteria loads, inflammatory cytokine levels, and pathological changes of tissues were determined. The results showed that PBD-2 (25-200 μg·mL^-1) could significantly inhibit the growth of S. suis SC-19 in a concentration-dependent manner (P<0.05). At the same time, PBD-2 had no significant toxic effect on RAW cells (P>0.05). The in vivo study revealed that PBD-2 treatment could effectively reduce the mortality of mice infected with S. suis SC-19. At the same time, PBD-2 treatment significantly reduced bacteria loads in brain, lung, spleen and blood of mice, and decreased the levels of inflammatory cytokines IL-6, IL-12 and TNF-α in mouse sera (P<0.05). At the same time, PBD-2 treatment also significantly alleviated the degree of pathological damages in lung and spleen tissues of mice infected with bacteria (P<0.05). These results indicate that PBD-2 confers great resilience to S. suis in vitro without significant cytotoxicity, while it also exhibits a therapeutic effect on S. suis infection in mice, suggesting that the use of PBD-2 as a therapeutic drug and substitutes for antibiotics is of an excellent prospect.     

High Throughput Sequencing to Study the Effect of Chronic Renal Failure on the Diversity and Gene Function Prediction of Gut Microbiota in Dogs

This study aimed to investigate the effect of chronic renal failure(CRF) on the gut microbiota diversity and predict the gene function of the flora. A total of 30 2-year-old dogs were selected and randomly divided into the chronic renal failure model group (CRF, established by renal artery ligation), sham operation control group (Sham) and healthy control group (HCG). All animals were fed normally during the 56 days of test period. The serum creatinine (Scr), blood urea nitrogen (BUN) and urine protein / creatinine ratio (UP/C) were detected regularly during the experiment. The effects of chronic renal failure on the structure, diversity and function of gut microbiota were analyzed according to the result of bacterial 16S rDNA sequencing from fresh feces collected without contamination. The flora markers index (FMI) was constructed based on the principal component Logistic regression analysis of different microbiota in CRF group to predict the development of chronic renal failure. The results showed that: 1) The levels of Scr, BUN and UP/C in CRF group from the start of the 28th day of the experiment were significantly higher than those of HCG group and Sham group (P<0.05), and significantly higher than that of the first day of CRF group (P<0.05). 2) Compared with those before chronic renal failure (CRF group at day 0 and 28), HCG group and Sham group, the Chao 1 diversity and Shannon diversity of gut microbiota in CRF group at day 56 were significantly lower (P<0.05), while the relative abundance of Bacteroidetes, Proteobacteria and Actinobacteria significantly increased and the number of Firmicutes significantly decreased (P<0.05). 3) LEfSe analysis showed that 20 species were enriched in CRF group, mainly including Pseudomonas, Escherichia, Proteus and so on, and most of them had negative correlation with other intestinal bacteria. Functional prediction revealed that genes of those different species were mainly enriched in amino acid metabolism, sugar biosynthesis and metabolism and carbohydrate metabolism in CRF group. The area under ROC curve (AUC) of the FMI constructed with those species enriched was 0.788, which could be used as the intestinal microbial marker for CRF in dogs. In summary, chronic renal failure can reduce the diversity of intestinal microbiota, lead to the imbalance of bacterial structure and change of bacterial function. Moreover, the enriched gut microbiota in CRF group can be used as the intestinal microbial marker of CRF in dogs, and the best prediction effect can be obtained by FMI.     

Antagonistic rhizoplane bacteria induce diverse morphological alterations in Peronosporomycete hyphae during <Emphasis Type="Italic">in vitro</Emphasis> interaction

A total of 150 bacteria were isolated from rhizoplanes of the host and non-host plants of a phytopathogenic Peronosporomycete Aphanomyces cochlioides. Upon screening, 5% of the isolates were evaluated as antagonists as they inhibited radial growth of A. cochlioides AC-5 hyphae in a dual culture assay. In addition, those antagonistic bacteria also induced characteristic morphological alterations in the A. cochlioides AC-5 hyphae that grew towards bacterial colonies. Hyphal morphological alterations observed in AC-5 and other tested strains of Peronosporomycetes included excessive branching, curly growth, unusually longer and pointed tip formation and swelling; all of these were comparable to the alterations induced by known antimicrobial compounds. Among the antagonistic bacteria, Pseudomonas sp. strain EC-S101 induced a unique branching pattern (tree-like) in AC-5 hyphae by continuous apical bifurcation of successive hyphae, where increases in number of branches and hyphal area were linearly correlated with time up to 10 h. Our observations suggested that the pathogen might have lost its ability of normal branch production; however maintained the capability of self-branching. Soluble extracts from the culture fluids of Pseudomonas sp. strain EC-S101 and Stenotrophomonas maltophilia EC-S105 induced similar excessive branching and curly growth in A. cochlioides hyphae as the respective bacterium. These results revealed that bacterial metabolites appeared to be responsible for induction of morphological alterations. Interestingly, the antagonistic bacteria that induced hyphal morphological alterations, also efficiently suppressed in vivo damping-off disease caused by AC-5. We suggest that antagonistic rhizoplane bacteria have the capability to induce diverse morphological alterations in Peronosporomycetes hyphae during in vitro interactions. Hyphal morphological alterations associated with growth inhibition and the induction of characteristic morphological changes indicate antagonistic activity against the Peronosporomycete.     

Classification and Genetic Diversity of Three Hare Species in Xinjiang Based on Mitochondrial DNA

The study aimed to explore the phylogeny and genetic diversity of 3 hare species in Xinjiang by molecular genetics methods, define the relationship and taxonomy status, assess diversity level of Lepus in Xinjiang, and provide the basic data for conservation genetics of hares in Xinjiang and even in China. Three mitochondrial DNA genes, COI, ND4 and 16S rRNA, were used as molecular markers, and the sequences of 3 genes of 57 samples collected from 8 different regions (4 geographic groups) in Xinjiang were determined by PCR amplification and sequencing technology. After the sequences of COI, ND4 and 16S rRNA of each sample were revised and pooled together, data were analyzed with softwares such as MEGA 7, DNAsp 6, Arlequin 3.1 and MrBayes 3.2. A total of 43 haplotypes were detected from the combined sequences of 3 genes of 57 hare samples. Five distinct clades (A-E) and 3 clusters were clearly showed in phylogenetic tree and median-joining network (MJN). Furthermore, the genetic distance between 3 clusters reached the level of species (4.21%-9.09%). However, the genetic distance between hares from northern Xinjiang (Clade E) and those from central Xinjiang (Clade D) were not up to the level of species (≤2.26%) in the third cluster. The haplotype diversity (h) of Lepus yarkandensis, Lepus tibetanus pamirensis and Lepus tolai lehmanni were higher(0.979±0.014, 0.972±0.064 and 0.972±0.064, respectively), while the nucleotide diversity (π) of the L. t. lehmanni and L. t. centrasiaticus were higher (0.033±0.018 and 0.023±0.015, respectively). Based on comprehensive analysis of 3 genes of mitochondrion and reference with published research, it is suggested that hares from southwestern Pamir Plateau of Xinjiang should belong to L. t. pamirensis. Meanwhile,hares distributed in northern and central Xinjiang might be considered as L. t. lehmanni and L. t. centrasiaticus. Moreover, there is abundant genetic diversity in the 3 hare species in Xinjiang, and the obvious phylogeographic pattern is showed.     

Recent observations on leafy gall in Liliaceae and some other families

Corynebacterium fascians, which causes leafy gall, has been shown to be responsible for the unusual symptoms recently found in lilies. The symptoms are described and the different bacterial isolates compared by biochemical, serological and pathogenicity experiments. No evidence could be found for the existence of specialized strains even though considerable variation in virulence could be demonstrated. Although the role of variation in susceptibility of the different lily cultivars should not be underestimated, it would appear that high inoculum levels ofC. fascians in the soil may be largely responsible for these outbreaks.Also included in this study are the results of biochemical, serological and pathogenicity experiments ofC. fascians which has been isolated fromKalanchoe, Euphorbia, Brodiaea, Hebe andVerbascum.Samenvatting Reeds een aantal jaren was in lelies een woekering in stengelbollen bekend, die echter sporadisch voorkwam en economisch van weinig betekenis was. Tijdens de zomer van 1977 echter trad de ziekte op één enkel perceel zo ernstig op dat zeer aanzienlijke oogstderving het gevolg was. Behalve de stengelbol kan ook de hoofdbol misvormd zijn. Bovendien kunnen misvormde stengelbollen lange spruiten vormen, zgn. sabeltandvorming. Soms is het wortelstelsel gereduceerd; in de kas zijn verdikte stengels waargenomen. De ziekte staat nu bekend onder de naam woekerziekte.Door de PD te Wageningen en het LBO te Lisse werd de bacterieCorynebacterium fascians (Tilford) Dows. uit het zieke materiaal geïsoleerd. In gezamenlijk onderzoek werd nagegaan of de bacterie de veroorzaker is van bovengenoemde symptomen en of hier sprake is van een op lelie gespecialiseerde stam.Biochemische en serologische vergelijkingen tussen isolaten vanC. fascians uit lelie,Kalanchoe, Euphorbia, Brodiaeea, Hebe enVerbascum laten zien dat er zeer weinig verschillen bestaan. Alle isolaten reageren op een standaard antiserum met titers variërend tussen 1: 640–1: 5120.Een oriënterende inoculatieproef werd uitgevoerd op het LBO in 1977. Daarna werden op de PD 10 van de 25 isolaten uit lelie en 8 uit andere waardplanten getoetst op lelie, chrysant en lathyrus. Uit de inoculatieproeven blijkt, dat de bacterie de veroorzaker is van de waargenomen symptomen. Tussen de isolaten blijkt een groot verschil in pathogeniteit te bestaan (12–100% aantasting), zonder dat dit aan herkomst gebonden is. Het voorkomen van op lelie of andere waardplanten gespecialiseerde stammen is daarom niet aan te nemen.In hoeverre de hevige aantasting een gevolg is van een opbouw van de bacterie-populatie door een te nauwe vruchtwisseling en mechanische rooimethoden moet worden nagegaan.     

Italian University System has to Match Educational Needs in Adherence to European Standards and Professional Scenario

The effect of a water-soluble fraction (WSF) of a non-pathogenic strain of Mycobacterium phlei was studied in bovine subclinical mastitis (SCM) by measuring the myeloperoxidase and acid phosphatase enzyme levels in the milk leukocytes. Forty-five cows were divided into three equal groups. Group I, consisting of 15 healthy cows, served as the control, whereas groups II and III each contained 15 cows with subclinical mastitis on the basis of a positive reaction in the California mastitis test (CMT). The cows in group II received 100 microg of WSF in 5 ml sterile phosphate-buffered saline, pH 7.4 (PBS) once only, while those in group III received 5 ml sterile PBS daily for 7 days, both treatments being given by the intramammary route. Observations were made up to 30 days after treatment (AT). The CMT of the healthy milk was negative (0), whereas it ranged between 1 and 2 points in SCM. The somatic cell count (SCC) increased significantly (p < 0.05) on day 3, then fell steeply from day 7 up to day 30 AT in the cows in group II. A steady decrease in the total bacterial count (TBC) was observed in the group treated with WSF but the bacterial counts remained high in the groups treated with PBS. The mean acid phosphatase level was enhanced by 119% on day 3 AT in group II but only by 18.7% in the cows in group III. The mean myeloperoxidase level was enhanced by 100% in the cows in group II but only by 18% in those in group III on day 3 AT. This significant reduction in the bacterial load in infected cows caused by intramammary infusion of WSF may be due to activation of the microbicidal activity of the neutrophils, but this requires confirmation.     

长期放牧对青藏高原高寒灌丛植被的影响

通过在青藏高原对放牧第18年的植物种多样性、群落结构、地上现存生物量和草场质量的研究结果表明随放牧率增加,植物种多样性指数的变化是一个典型的单峰曲线模式.长期重度放牧使高寒灌丛群落结构简化,地上现存生物量特别是优良牧草的现存量减少.植物群落的高度、总盖度和枯草盖度随着放牧强度的降低而增加,绿色植物的盖度在中度放牧样地最高.从轻牧到重牧,灌木和禾草的优势地位被典型杂类草替代.长期重度放牧在青藏高原草场退化过程中起重要作用,在青藏高原实施"取半留半"的放牧原则,对于防止草场退化,提高牧草利用率,维持较高的生物多样性都有益处.     

Genetic Diversity and Relationships of Japanese Peach (<Emphasis Type="Italic">Prunus persica</Emphasis> L.) Cultivars Revealed by AFLP and Pedigree Tracing

To evaluate the genetic diversity and to clarify the genetic relationships of Japanese peach cultivars, we analyzed the amplified fragment length polymorphism (AFLP) and traced the pedigree of 17 Japanese commercial peach cultivars and six traditional accessions. Sixteen AFLP primer combinations produced a total of 837 fragments and 146 polymorphic bands with a polymorphism percentage of 17.5%. All of the peach accessions could be identified from differences in at least 10 polymorphic bands. A cluster analysis showed that all the Japanese commercial peach cultivars, except ‘Kiyomi’ and ‘Jichigetsuto’, formed a major group consisting of three sub-groups. Of the six traditional accessions, four were genetically distant from the Japanese commercial peach cultivars while two accessions from China were classified into the Japanese commercial peach cultivars group. Both the AFLP analysis and pedigree tracing suggested that Japanese commercial peach cultivars are mainly derived from ‘Shanhai Suimitsuto’, one of the traditional accessions from China. Although the genetic relationships revealed by AFLP were generally in agreement with those shown by the pedigree information, some contradictions were found. Combining the AFLP results and pedigree information can provide a better understanding of the genetic relationships of Japanese peach cultivars.