Teleconnections between large-scale oceanic-atmospheric patterns and interannual surface wind speed variability across China: Regional and seasonal patterns.

Great attention has been paid to the long-term decline in terrestrial near-surface wind speed (SWS) in China. However, how the SWS varies with regions and seasons and what modulates these changes remain unclear. Based on quality-controlled and homogenized terrestrial SWS data from 596 stations, the covarying SWS patterns during the Asian Summer Monsoon (ASM) and the Asian Winter Monsoon (AWM) seasons are defined for China using empirical orthogonal function (EOF) analysis for 1961-2016. The dominant SWS features represented by EOF1 patterns in both seasons show a clear decline over most regions of China. The interannual variability of the EOF1 patterns is closely related to the Northeast Asia Low Pressure (NEALP) and the Arctic Oscillation (AO), respectively. The EOF2 and EOF3 patterns during ASM (AWM) season describe a dipole mode of SWS between East Tibetan Plateau and East China Plain (between East Tibetan Plateau and Northeast China), and between Southeast and Northeast China (between Northeast China and the coastal areas of Southeast China), respectively. These dipole structures of SWS changes are closely linked with the oceanic-atmospheric oscillations on interannual scale.

¿Hay mejores parámetros diagnósticos de la luteinización precoz que el valor aislado de los niveles de progesterona o el cociente progesterona/es-tradiol el día de administración de hCG? / There are better parameters Diagnostics for premature luteinization thanisolate value of Progesterone level or progesterone/estradiol ratio on theday of hCG administration?

Objetivo: Tanto el valor absoluto de progesterona (P) como el cociente progesterona/estradiol (P/E)son utilizados para el diagnóstico de la luteinización precoz (LP), sin llegar a un acuerdo en el umbraladecuado y con resultados muy dispares en cuanto a su capacidad predictiva. El cociente P/E ha creadoademás una gran confusión entre el concepto de LP y la baja respuesta ovárica a la hiperestimulación.La única alternativa viable es encontrar un parámetro que resulte independiente del grado de respuestaovárica y los valores de estradiol.Método:Se analizaron 434 ciclos consecutivos de FIV con estimulación mediante FSH y/o LH y fre-nado hipofisario con agonistas o antagonistas de GnRH sin ningún tipo de criterio de exclusión previopara determinar la relación matemática existente entre progesterona y estradiol en el momento previoa la ovulación.Resultados:La relación empírica entre progesterona y estradiol viene establecida por la fórmula E=4P2,de la que podemos deducir las fórmulas descriptivas de P=0,5E0,5, P/E=0,5E-0,5 y P2/E=0,25, siendoP2/E la única que corresponde a una constante sin dependencia del nivel de estradiol y, por tanto, el cri-terio ideal para el diagnóstico de LP. (AU)

Introduction: Both the absolute value of progesterone (P) and the progesterone/estradiol ratio (P/E) are used for the diag-nosis of early luteinization (EL), without reaching an agreement on the appropriate threshold and with very differentresults in terms of its predictive ability. The P/E ratio has also created a great deal of confusion between the concept of LPand low ovarian response to hyperstimulation. The objective of this study is to find a parameter that is independent of the degree of ovarian response and estradiol va-lues.Methods: We analyzed 434 consecutive IVF cycles with FSH and/or LH stimulation and pituitary down regulation withagonist or antagonists without any prior exclusion criteria to determine the mathematical relationship between progesteroneand estradiol at the time prior to ovulation.Results: The empirical relationship between progesterone and estradiol is established by the formula E=4P2, from whichwe can deduce the descriptive formulas of P=0,5E0,5, P/E=0,5E-0,5 an P2/E=0,25, being P2/E the only one that corres-ponds to a constant without dependence on the estradiol level and, therefore, the ideal criterion for the diagnosis of early. (AU)

In Vivo Bioluminescent Imaging of Yersinia ruckeri Pathogenesis in Fish.

Bioluminescent reporters and advanced luciferase technologies are useful to study host-pathogen interactions. This chapter describes the use of the luxCDABE operon from Photorhabdus luminescens as a tool to analyze the progression of the fish pathogen Yersinia ruckeri during the infection of rainbow trout, as well as the quantification of promoter activity of specific bacterial genes during host colonization.

Changes in physiology and virulence during the selection of resistant Yersinia ruckeri mutants under subinhibitory cefotaxime concentrations.

Bacterial antibiotic resistance is one of the main healthcare problems currently. Apart from reducing antibiotic efficacy, it has awakened the interest of scientists due to its association with bacterial fitness and virulence. Interestingly, antibiotic resistance can be a source of both increased fitness and decreased fitness, even though the molecular basis of these relationships remains unknown. The aim of this work is to define the effects of sub-MIC concentrations of cefotaxime, an antibiotic extensively used in clinical practice, on the physiology and virulence of Yersinia ruckeri and to determine the importance of these sub-MIC concentrations for the selection of antibiotic-resistant mutants in the aquatic environment. Results indicated that exposure to sub-MIC concentrations of cefotaxime selected Y. ruckeri populations with irreversible alterations in the physiology, such as slow growth, aggregation in liquid cultures and modification of the colony morphology. These bacteria also displayed changes in the OMPs and LPS profiles and a full attenuation of virulence. An overexpression of the envelope stress regulator RpoE was also detected after exposure to the antibiotic. In conclusion, exposure to cefotaxime selected, at high frequency, Y. ruckeri strains that survive the antibiotic stress at the expense of a fitness cost and the loss of virulence.

The Infection Process of Yersinia ruckeri: Reviewing the Pieces of the Jigsaw Puzzle.

Finding the keys to understanding the infectious process of Yersinia ruckeri was not a priority for many years due to the prompt development of an effective biotype 1 vaccine which was used mainly in Europe and USA. However, the gradual emergence of outbreaks in vaccinated fish, which have been reported since 2003, has awakened interest in the mechanism of virulence in this pathogen. Thus, during the last two decades, a large number of studies have considerably enriched our knowledge of many aspects of the pathogen and its interaction with the host. By means of both conventional and a variety of novel strategies, such as cell GFP tagging, bioluminescence imaging and optical projection tomography, it has been possible to determine three putative Y. ruckeri infection routes, the main point of entry for the bacterium being the gill lamellae. Moreover, a wide range of potential virulence factors have been highlighted by specific gene mutagenesis strategies or genome-wide transposon/plasmid insertion-based screening approaches, such us in vivo expression technology (IVET) and signature tagged mutagenesis (STM). Finally, recent proteomic and whole genomic analyses have allowed many of the genes and systems that are potentially implicated in the organism's pathogenicity and its adaptation to the host environmental conditions to be elucidated. Altogether, these studies contribute to a better understanding of the infectious process of Y. ruckeri in fish, which is crucial for the development of more effective strategies for preventing or treating enteric redmouth disease (ERM).

Temperature-Dependent Gene Expression in Yersinia ruckeri: Tracking Specific Genes by Bioluminescence During in Vivo Colonization.

Yersinia ruckeri is a bacterium causing fish infection processes at temperatures below the optimum for growth. A derivative Tn5 transposon was used to construct a library of Y. ruckeri mutants with transcriptional fusions between the interrupted genes and the promoterless luxCDABE and lacZY operons. In vitro analysis of ß-galactosidase activity allowed the identification of 168 clones having higher expression at 18°C than at 28°C. Among the interrupted genes a SAM-dependent methyltransferase, a diguanylated cyclase, three genes involved in legionaminic acid synthesis and three transcriptional regulators were defined. In order to determine, via bioluminescence emission, the in vivo expression of some of these genes, two of the selected mutants were studied. In one of them, the acrR gene coding a repressor involved in regulation of the AcrAB-TolC expulsion pump was interrupted. This mutant was found to be highly resistant to compounds such as chloramphenicol, tetracycline, and ciprofloxacin. Although acrR mutation was not related to virulence in Y. ruckeri, this mutant was useful to analyze acrR expression in fish tissues in vivo. The other gene studied was osmY which is activated under osmotic stress and is involved in virulence. In this case, complemented mutant was used for experiments with fish. In vivo analysis of bioluminescence emission by these two strains showed higher values for acrR in gut, liver and adipose tissue, whereas osmY showed higher luminescence in gut and, at the end of the infection process, in muscle tissue. Similar results were obtained in ex vivo assays using rainbow trout tissues. The results indicated that this kind of approach was useful for the identification of genes related to virulence in Y. ruckeri and also for the in vivo and in vitro studies of each of the selected genes.

More Than Gliding: Involvement of GldD and GldG in the Virulence of Flavobacterium psychrophilum.

A fascinating characteristic of most members of the genus Flavobacterium is their ability to move over surfaces by gliding motility. Flavobacterium psychrophilum, an important pathogen of farmed salmonids worldwide, contains in its genome the 19 gld and spr genes shown to be required for gliding or spreading in Flavobacterium johnsoniae; however, their relative role in its lifestyle remains unknown. In order to address this issue, two spreading deficient mutants were produced as part of a Tn4351 mutant library in F. psychrophilum strain THCO2-90. The transposons were inserted in gldD and gldG genes. While the wild-type strain is proficient in adhesion, biofilm formation and displays strong proteolytic activity, both mutants lost these characteristics. Extracellular proteome comparisons revealed important modifications for both mutants, with a significant reduction of the amounts of proteins likely transported through the outer membrane by the Type IX secretion system, indicating that GldD and GldG proteins are required for an effective activity of this system. In addition, a significant decrease in virulence was observed using rainbow trout bath and injection infection models. Our results reveal additional roles of gldD and gldG genes that are likely of importance for the F. psychrophilum lifestyle, including virulence.

Comparative genome analysis reveals important genetic differences among serotype O1 and serotype O2 strains of Y. ruckeri and provides insights into host adaptation and virulence.

Despite the existence of a commercial vaccine routinely used to protect salmonids against Yersinia ruckeri, outbreaks still occur, mainly caused by nonmotile and lipase-negative strains (serotype O1 biotype 2). Moreover, epizootics caused by other uncommon serotypes have also been reported. At the moment, one of the main concerns for the aquaculture industry is the expanding range of hosts of this pathogen and the emergence of new biotypes and serotypes causing mortality in fish farms and against which the vaccine cannot protect. The comparative analysis of the genome sequences of five Y. ruckeri strains (150, CSF007-82, ATCC29473, Big Creek 74, and SC09) isolated from different hosts and classified into different serotypes revealed important genetic differences between the genomes analyzed. Thus, a clear genetic differentiation was found between serotype O1 and O2 strains. The presence of 99 unique genes in Big Creek 74 and 261 in SC09 could explain the adaptation of these strains to salmon and catfish, respectively. Finally, the absence of 21 genes in ATCC29473 which are present in the other four virulent strains could underpin the attenuation described for this strain. The study reveals important genetic differences among the genomes analyzed. Further investigation of the genes highlighted in this study could provide insights into the understanding of the virulence and niche adaptive mechanisms of Y. ruckeri.

Complete Genome Sequence of Flavobacteriumpsychrophilum Strain OSU THCO2-90, Used for Functional Genetic Analysis.

We report here the complete annotated genome sequence of Flavobacterium psychrophilum OSU THCO2-90, isolated from Coho salmon (Oncorhynchus kisutch) in Oregon. The genome consists of a circular chromosome with 2,343 predicted open reading frames. This strain has proved to be a valuable tool for functional genomics.

Genome Sequence of the Fish Pathogen Yersinia ruckeri Strain 150, Isolated from Diseased Rainbow Trout.

We present here the draft genome of a pathogenic Yersinia ruckeri strain, isolated from rainbow trout (Oncorhynchus mykiss) affected by enteric redmouth disease. The chromosome has 3,826,775 bp, a GC content of 46.88%, and is predicted to contain 3,538 coding sequences. The data will be useful for comparative pathogenicity studies.

Temperature-dependent expression of virulence genes in fish-pathogenic bacteria.

Virulence gene expression in pathogenic bacteria is modulated by environmental parameters. A key factor in this expression is temperature. Its effect on virulence gene expression in bacteria infecting warm-blooded hosts is well documented. Transcription of virulence genes in these bacteria is induced upon a shift from low environmental to a higher host temperature (37°C). Interestingly, host temperatures usually correspond to the optimum for growth of these pathogenic bacteria. On the contrary, in ectothermic hosts such as fish, molluscs, and amphibians, infection processes generally occur at a temperature lower than that for the optimal growth of the bacteria. Therefore, regulation of virulence gene expression in response to temperature shift has to be modulated in a different way to that which is found in bacteria infecting warm-blooded hosts. The current understanding of virulence gene expression and its regulation in response to temperature in fish-pathogenic bacteria is limited, but constant extension of our knowledge base is essential to enable a rational approach to the problem of the bacterial fish diseases affecting the aquaculture industry. This is an interesting issue and progress needs to be made in order to diminish the economic losses caused by these diseases. The intention of this review is, for the first time, to compile the scattered results existing in the field in order to lay the groundwork for future research. This article is an overview of those relevant virulence genes that are expressed at temperatures lower than that for optimal bacterial growth in different fish-pathogenic bacteria as well as the principal mechanisms that could be involved in their regulation.

Development of a markerless deletion system for the fish-pathogenic bacterium Flavobacterium psychrophilum.

Flavobacterium psychrophilum is a Gram-negative fish pathogen that causes important economic losses in aquaculture worldwide. Although the genome of this bacterium has been determined, the function and relative importance of genes in relation to virulence remain to be established. To investigate their respective contribution to the bacterial pathogenesis, effective tools for gene inactivation are required. In the present study, a markerless gene deletion system has been successfully developed for the first time in this bacterium. Using this method, the F. psychrophilum fcpB gene, encoding a predicted cysteine protease homologous to Streptococcus pyogenes streptopain, was deleted. The developed system involved the construction of a conjugative plasmid that harbors the flanking sequences of the fcpB gene and an I-SceI meganuclease restriction site. Once this plasmid was integrated in the genome by homologous recombination, the merodiploid was resolved by the introduction of a plasmid expressing I-SceI under the control of the fpp2 F. psychrophilum inducible promoter. The resulting deleted fcpB mutant presented a decrease in extracellular proteolytic activity compared to the parental strain. However, there were not significant differences between their LD50 in an intramuscularly challenged rainbow trout infection model. The mutagenesis approach developed in this work represents an improvement over the gene inactivation tools existing hitherto for this "fastidious" bacterium. Unlike transposon mutagenesis and gene disruption, gene markerless deletion has less potential for polar effects and allows the mutation of virtually any non-essential gene or gene clusters.

Lack of a type-2 glycosyltransferase in the fish pathogen Flavobacterium psychrophilum determines pleiotropic changes and loss of virulence.

Flavobacterium psychrophilum is an important fish pathogen, responsible for Cold Water Disease, with a significant economic impact on salmonid farms worldwide. In spite of this, little is known about the bacterial physiology and pathogenesis mechanisms, maybe because it is difficult to manipulate, being considered a fastidious microorganism. Mutants obtained using a Tn4351 transposon were screened in order to identify those with alteration in colony morphology, colony spreading and extracellular proteolytic activity, amongst other phenotypes. A F. psychrophilum mutant lacking gliding motility showed interruption of the FP1638 locus that encodes a putative type-2 glycosyltransferase (from here on referred to as fpgA gene, Flavobacterium psychrophilum glycosyltransferase). Additionally, the mutant also showed a decrease in the extracellular proteolytic activity as a consequence of down regulation in the fpgA mutant background of the fpp2-fpp1 operon promoter, responsible for the major extracellular proteolytic activity of the bacterium. The protein glycosylation profile of the parental strain showed the presence of a 22 kDa glycosylated protein which is lost in the mutant. Complementation with the fpgA gene led to the recovery of the wild-type phenotype. LD50 experiments in the rainbow trout infection model show that the mutant was highly attenuated. The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium. Moreover, the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine.

The heat sensitive factor (HSF) of Yersinia ruckeri is produced by an alkyl sulphatase involved in sodium dodecyl sulphate (SDS) degradation but not in virulence.

BACKGROUND: The heat sensitive factor (HSF) of the fish pathogen Yersinia ruckeri was previously identified as an unusual band on SDS-PAGE. According to this, Y. ruckeri strains were classified in HSF+ and HSF - in terms of the presence/absence of the factor. Experiments carried out by injection challenge with HSF + strains caused high mortalities in rainbow trout. In contrast, HSF - strains did not cause mortality. In conclusion, HSF appeared to be a relevant virulence factor in Y. ruckeri. RESULTS: We report here the identification and study of the gene coding for the enzyme involved in the production of HSF. Culture medium containing SDS and Coomassie brilliant blue dye was used to screen a mini-Tn5 Km2 mutant library of Y. ruckeri 150. Blue colonies lacking a surrounding creamy deposit, a phenotype described in former studies as HSF - , were identified. DNA sequence analysis of a selected mutant revealed that this had a transposon interruption in a chromosome-located gene which codes for a heat sensitive alkyl sulphatase of 78.7 kDa (YraS; Yersinia ruckeri alkyl sulphatase) which is able to degrade SDS to 1-dodecanol. As it was expected, the introduction of the yraS gene into an HSF - strain turned this into HSF + . Surprisingly, although the protein allows Y. ruckeri to degrade SDS, the bacterium could not use this compound as the sole carbon source. Moreover, the yraS mutant showed a similar level of SDS resistance to the parental strain. It was the interruption of the acrA gene which made Y. ruckeri sensitive to this compound. LD50 experiments showed a similar virulence of the yraS mutant and parental strain. CONCLUSIONS: The HSF of Y. ruckeri is the product of the alkyl sulphatase YraS, able to degrade SDS to 1-dodecanol. This degradation is not linked to the utilization of SDS as a carbon source and surprisingly, the enzyme is not involved in bacterial virulence or in the high SDS resistance displayed by the bacterium. This role is played by the AcrAB-TolC system.

Flavobacterium psychrophilum vaccine development: a difficult task.

Bacterial cold water disease (BCWD) is a globally distributed freshwater fish disease caused by the Gram-negative bacterium Flavobacterium psychrophilum. It is a particularly devastating infection in fry salmonids and may lead to high levels of mortality. In spite of its economic impact on fish farms, neither the biology of the bacterium nor the bacterium-host interactions are well understood. This review provides a synopsis of the major problems related to critical remaining questions about research into the use of vaccines against F. psychrophilum and the development of a commercial vaccine against this disease. Studies using sera from convalescent rainbow trout have shown the antigenic properties of different proteins such as OmpH, OmpA and FspA, as well as low and high molecular mass lipopolysaccharide of F. psychrophilum, which are potential candidates for subunit vaccines. Inactivated F. psychrophilum bacterins have been successfully tested as vaccines under laboratory conditions by both immersion and intraperitoneal routes. However, the efficacy and the practical usefulness of these preparations still have to be proved. The use of attenuated and wild-type strains to immunize fish showed that these systems offer high levels of protection. Nevertheless, their application clashes with the regulations for environmental protection in many countries. In conclusion, protective vaccines against BCWD are theoretically possible, but substantial efforts still have to be made in order to permit the development of a commercial vaccine.

The yrpAB operon of Yersinia ruckeri encoding two putative U32 peptidases is involved in virulence and induced under microaerobic conditions.

In an attempt to dissect the virulence mechanisms of Yersinia ruckeri two adjacent genes, yrpA and yrpB, encoding putative peptidases belonging to the U32 family, were analyzed. Similar genes, with the same genetic organization were identified in genomic analysis of human-pathogenic yersiniae. RT-PCR studies indicated that these genes form an operon in Y. ruckeri. Transcriptional studies using an yrpB::lacZY fusion showed high levels of expression of these genes in the presence of peptone in the culture medium, as well as under oxygen-limited conditions. These two factors had a synergic effect on gene induction when both were present simultaneously during bacterial incubation, which indicates the important role that environmental conditions in the fish gut can play in the regulation of specific genes. LD 50 experiments using an yrpA insertional mutant strain demonstrated the participation of this gene in the virulence of Y. ruckeri.

Genome sequence of Lactococcus garvieae IPLA 31405, a bacteriocin-producing, tetracycline-resistant strain isolated from a raw-milk cheese.

This work describes the draft genome sequence of Lactococcus garvieae IPLA 31405, isolated from a traditional Spanish cheese. The genome contains a lactose-galactose operon, a bacteriocin locus, two integrated phages, a transposon harboring an active tet(M) gene, and two theta-type plasmid replicons. Genes encoding virulence factors were not recorded.

Construction and validation of a GFP-based vector for promoter expression analysis in the fish pathogen Flavobacterium psychrophilum.

The study of the fish pathogen Flavobacterium psychrophilum has been drastically hampered by the difficulty to perform genetic manipulation of this organism. Although recent publications described the successful transfer of genetic material into this bacterium by transformation and conjugation, additional tools are still needed. This paper reports the construction of vector pCP23-G, which permits for the first time to monitor transcriptional regulation in this pathogen by using a promoterless gfpmut3 gene as a reporter. Additionally, use of pCP23-G enabled the trancriptional analysis of three putative promoter regions of F. psychrophilum, corresponding to genes fpp2-fpp1, pdhB and gldJ, under different growth conditions. Overall, the construction of pCP23-G facilitates genetic analysis in F. psychrophilum, by enabling the determination of gene expression both in vitro and in vivo. Furthermore, this would also open the possibility for studies on the location of this bacterium in the fish tissues.

Genome sequence of Lactococcus garvieae UNIUD074, isolated in Italy from a lactococcosis outbreak.

Lactococcus garvieae is the etiological agent of lactococcosis disease, affecting many cultured fish species worldwide. In addition, this bacterium is currently considered a potential zoonotic microorganism since it is known to cause several opportunistic human infections. Here we present the draft genome sequence of the L. garvieae strain UNIUD074.

Application of suppressive subtractive hybridization to the identification of genetic differences between two Lactococcus garvieae strains showing distinct differences in virulence for rainbow trout and mouse.

Lactococcus garvieae is the causative microbial agent of lactococcosis, an important and damaging fish disease in aquaculture. This bacterium has also been isolated from vegetables, milk, cheese, meat and sausages, from cow and buffalo as a mastitis agent, and even from humans, as an opportunistic infectious agent. In this work pathogenicity experiments were performed in rainbow trout and mouse models with strains isolated from human (L. garvieae HF) and rainbow trout (L. garvieae UNIUDO74; henceforth referred to as 074). The mean LD(50) value in rainbow trout obtained for strain 074 was 2.1 × 10(2) ± 84 per fish. High doses of the bacteria caused specific signs of disease as well as histological alterations in mice. In contrast, strain HF did not prove to be pathogenic either for rainbow trout or for mice. Based on these virulence differences, two suppressive subtractive hybridizations were carried out to identify unique genetic sequences present in L. garvieae HF (SSHI) and L. garvieae 074 (SSHII). Differential dot-blot screening of the subtracted libraries allowed the identification of 26 and 13 putative ORFs specific for L. garvieae HF and L. garvieae 074, respectively. Additionally, a PCR-based screening of 12 of the 26 HF-specific putative ORFs and the 13 074-specific ones was conducted to identify their presence/absence in 25 L. garvieae strains isolated from different origins and geographical areas. This study demonstrates the existence of genetic heterogeneity within L. garvieae isolates and provides a more complete picture of the genetic background of this bacterium.